Journal articles on the topic 'Amplicon sequence variant'
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Liao, Yu-Chieh, Feng-Jui Chen, Min-Chieh Chuang, Han-Chieh Wu, Wan-Chen Ji, Guann-Yi Yu, and Tsi-Shu Huang. "High-Integrity Sequencing of Spike Gene for SARS-CoV-2 Variant Determination." International Journal of Molecular Sciences 23, no. 6 (March 17, 2022): 3257. http://dx.doi.org/10.3390/ijms23063257.
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For tiling of the SARS-CoV-2 genome, the ARTIC Network provided a V4 protocol using 99 pairs of primers for amplicon production and is currently the widely used amplicon-based approach. However, this technique has regions of low sequence coverage and is labour-, time-, and cost-intensive. Moreover, it requires 14 pairs of primers in two separate PCRs to obtain spike gene sequences. To overcome these disadvantages, we proposed a single PCR to efficiently detect spike gene mutations. We proposed a bioinformatic protocol that can process FASTQ reads into spike gene consensus sequences to accurately call spike protein variants from sequenced samples or to fairly express the cases of missing amplicons. We evaluated the in silico detection rate of primer sets that yield amplicon sizes of 400, 1200, and 2500 bp for spike gene sequencing of SARS-CoV-2 to be 59.49, 76.19, and 92.20%, respectively. The in silico detection rate of our proposed single PCR primers was 97.07%. We demonstrated the robustness of our analytical protocol against 3000 Oxford Nanopore sequencing runs of distinct datasets, thus ensuring high-integrity sequencing of spike genes for variant SARS-CoV-2 determination. Our protocol works well with the data yielded from versatile primer designs, making it easy to determine spike protein variants.
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Darville-O'Quinn, Paige, Nalan Gokgoz, Kim M. Tsoi, Jay S. Wunder, and Irene L. Andrulis. "Abstract 5112: Investigating the use of circulating tumor DNA for sarcoma management." Cancer Research 82, no. 12_Supplement (June 15, 2022): 5112. http://dx.doi.org/10.1158/1538-7445.am2022-5112.
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Abstract Circulating tumor DNA (ctDNA) has the potential to detect sarcoma recurrence and metastasis but requires highly sensitive methods to detect and quantify genetic variants present in very low quantities. Plasma was isolated from 20mL peripheral blood samples collected from over 400 pre-operative sarcoma patients, and matched tumor samples from surgical resection were frozen and stored. Cell-free DNA (cfDNA) extracted from plasma was quantified using qPCR, and the quality was assessed using capillary electrophoresis. A subset of these cases were selected for whole exome sequencing (WES). WES of bulk tumor and whole blood samples identified tumor-specific genetic alterations, which then serve as personalized biomarkers of tumor DNA in patient plasma. We previously showed that droplet digital PCR (ddPCR) can detect and quantify ctDNA, by targeting patient-specific variants. However, ddPCR is limited in that it can only investigate one tumor variant sequence at a time. The purpose of the present study is to investigate methods of targeting multiple tumor variants simultaneously, increasing the chances of detecting ctDNA in patient blood. To this end, four cases were selected for multiplex PCR (mPCR) followed by targeted amplicon sequencing. For each case, six to eight of the tumor variants identified by WES were selected as targets, and primers were designed to amplify these sequences concurrently by mPCR. The amplicons will then be sequenced to detect the tumor variants. Additionally, two of the four cases have plasma collected at two different time points. To assess the viability of this method as a way to monitor disease surveillance, these cfDNA samples will be compared to determine how the abundance and nature of ctDNA changes over time. To date, cfDNA has been extracted from over 100 cases, the majority of which were positive for cfDNA. For each of the cases whole exome sequenced, a variety of tumor-specific variations were identified. The variants chosen as targets were selected based on having the highest variant allele frequency (VAF), with priority being given to mutations that alter the protein coding sequence. Thus far, mPCR primers have been designed and optimized for four separate cases. Across all cases analyzed by amplicon sequencing, the variant sequences could be detected in the amplicons generated by mPCR of tumor DNA. Furthermore, amplicon sequencing was able to recapitulate the variant allele frequency observed in WES. This indicates that the mPCR successfully amplified the sequences of interest in the tumor DNA, and that the sequencing results are accurate. Furthermore, no tumor variants were detected in the amplicons generated from blood DNA, which is to be expected. The cfDNA amplicons for these cases will be sequenced in this manner to investigate the presence of ctDNA. If successful, the ability to detect ctDNA in plasma will be an important first step in developing a testing protocol for clinical use. Citation Format: Paige Darville-O'Quinn, Nalan Gokgoz, Kim M. Tsoi, Jay S. Wunder, Irene L. Andrulis. Investigating the use of circulating tumor DNA for sarcoma management [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5112.
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Brandt, David, Marina Simunovic, Tobias Busche, Markus Haak, Peter Belmann, Sebastian Jünemann, Tizian Schulz, et al. "Multiple Occurrences of a 168-Nucleotide Deletion in SARS-CoV-2 ORF8, Unnoticed by Standard Amplicon Sequencing and Variant Calling Pipelines." Viruses 13, no. 9 (September 18, 2021): 1870. http://dx.doi.org/10.3390/v13091870.
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Genomic surveillance of the SARS-CoV-2 pandemic is crucial and mainly achieved by amplicon sequencing protocols. Overlapping tiled-amplicons are generated to establish contiguous SARS-CoV-2 genome sequences, which enable the precise resolution of infection chains and outbreaks. We investigated a SARS-CoV-2 outbreak in a local hospital and used nanopore sequencing with a modified ARTIC protocol employing 1200 bp long amplicons. We detected a long deletion of 168 nucleotides in the ORF8 gene in 76 samples from the hospital outbreak. This deletion is difficult to identify with the classical amplicon sequencing procedures since it removes two amplicon primer-binding sites. We analyzed public SARS-CoV-2 sequences and sequencing read data from ENA and identified the same deletion in over 100 genomes belonging to different lineages of SARS-CoV-2, pointing to a mutation hotspot or to positive selection. In almost all cases, the deletion was not represented in the virus genome sequence after consensus building. Additionally, further database searches point to other deletions in the ORF8 coding region that have never been reported by the standard data analysis pipelines. These findings and the fact that ORF8 is especially prone to deletions, make a clear case for the urgent necessity of public availability of the raw data for this and other large deletions that might change the physiology of the virus towards endemism.
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Gundry, Cameron N., Joshua G. Vandersteen, Gudrun H. Reed, Robert J. Pryor, Jian Chen, and Carl T. Wittwer. "Amplicon Melting Analysis with Labeled Primers: A Closed-Tube Method for Differentiating Homozygotes and Heterozygotes." Clinical Chemistry 49, no. 3 (March 1, 2003): 396–406. http://dx.doi.org/10.1373/49.3.396.
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Abstract Background: Common methods for identification of DNA sequence variants use gel electrophoresis or column separation after PCR. Methods: We developed a method for sequence variant analysis requiring only PCR and amplicon melting analysis. One of the PCR primers was fluorescently labeled. After PCR, the melting transition of the amplicon was monitored by high-resolution melting analysis. Different homozygotes were distinguished by amplicon melting temperature (Tm). Heterozygotes were identified by low-temperature melting of heteroduplexes, which broadened the overall melting transition. In both cases, melting analysis required ∼1 min and no sample processing was needed after PCR. Results: Polymorphisms in the HTR2A (T102C), β-globin [hemoglobin (Hb) S, C, and E], and cystic fibrosis (F508del, F508C, I507del, I506V) genes were analyzed. Heteroduplexes produced by amplification of heterozygous DNA were best detected by rapid cooling (>2 °C/s) of denatured products, followed by rapid heating during melting analysis (0.2–0.4 °C/s). Heterozygotes were distinguished from homozygotes by a broader melting transition, and each heterozygote had a uniquely shaped fluorescent melting curve. All homozygotes tested were distinguished from each other, including Hb AA and Hb SS, which differed in Tm by <0.2 °C. The amplicons varied in length from 44 to 304 bp. In place of one labeled and one unlabeled primer, a generic fluorescent oligonucleotide could be used if a 5′ tail of identical sequence was added to one of the two unlabeled primers. Conclusion: High-resolution melting analysis of PCR products amplified with labeled primers can identify both heterozygous and homozygous sequence variants.
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Kohlmann, Alexander, Hans-Ulrich Klein, Silvia Bresolin, Tracy Chaplin, Harry Cuppens, Bernardo Garicochea, Vera Grossmann, et al. "The Interlaboratory RObustness of Next-Generation Sequencing (IRON) Study: Deep-Sequencing Investigating TET2, CBL, and KRAS Mutations In 4464 Amplicons by An International Group Involving 8 Laboratories." Blood 116, no. 21 (November 19, 2010): 1665. http://dx.doi.org/10.1182/blood.v116.21.1665.1665.
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Abstract Abstract 1665 Massively parallel pyrosequencing in picoliter-sized wells is an innovative technique and allows highly-sensitive deep-sequencing to detect molecular aberrations. Thus far, limited data is available on the technical performance in a clinical diagnostic setting. Here, we investigated - as an international consortium - the robustness, precision, and reproducibility of 454 amplicon next-generation sequencing (NGS) across 8 laboratories from 6 countries. As a first candidate gene we selected TET2, a frequently mutated gene in myeloproliferative neoplasms. In total, 31 primer pairs including a 10-base molecular barcode sequence were designed and evaluated: All coding exons of TET2 were represented by 27 amplicons. In addition, 2 primer pairs were amplifying hotspot regions to characterize the RING finger domain and linker sequence for CBL and 2 amplicons covered KRAS exons 2 and 3. To execute our study, we used the small volume Titanium emulsion PCR setup (454 Life Sciences, Branford, CT). A cohort of 18 chronic myelomonocytic leukemia (CMML) patient samples were centrally collected by the Munich Leukemia Laboratory and characterized by conventional sequencing for mutations in TET2, CBL, and KRAS. In this selected cohort 33 distinct mutations in TET2, 7 mutations in CBL, and 3 mutations in KRAS, respectively, were detected by Sanger sequencing (plus 10 SNPs and one silent mutation). Each of the participating laboratories received anonymized aliquots of 1.6 μg of genomic DNA to be processed for the generation of PCR amplicons suitable for 454 deep-sequencing. In detail, a total of 31 × 18 (n=558) PCRs were locally performed at each laboratory, i.e. a total of 4464 PCR reactions across 8 centers. Subsequently, at each site each PCR product was individually purified and quantified and corresponding pools were generated by combining 31 amplicons in an equimolar ratio for each patient sample. After processing the samples using the 454 workflow, 3 patients each were loaded per lane on an 8-lane PicoTiterPlate on the GS FLX sequencer instrument. Overall, each of the 8 participating laboratories generated in median 432,606 reads across the 31 PCR amplicons (“Passed Filter Wells”). The median coverage per amplicon was 713-fold, ranging from 553-fold to 878-fold. Dropouts of single amplicons with no coverage obtained were observed in 4/8 laboratories in 61 of 4464 PCR products (1.4%). After alignment of the obtained sequences against the reference genome a total of 92 variants (44 distinct mutations and 10 SNPs) were observed across 22 amplicons. For this analysis, a given variant was scored if, in median, both forward and reverse reads were harboring the variant in at least 20% of reads, i.e. in line with the Sanger sequencing detection limit (GS FLX Amplicon Variant Analysis software v.2.3). In comparison to data available from Sanger sequencing, 454 amplicon deep-sequencing detected all mutations and SNPs that were previously known (few comparisons not possible due to single amplicon dropouts). In 90/92 variant comparisons all eight laboratories consistently detected the variant (two KRAS mutations being detected with a range from 18.0% - 22.6% of reads carrying the mutation). We did not observe a considerable bias in the measurements of the 92 variants between any two centers. Based on paired t-tests for equivalence, with equivalence limits for the standardized expected differences between two centers of -+ε (ε=0.5), the null hypothesis of dissimilar measurements was rejected for all pairs of centers (alpha=0.05). The estimated standard deviation of the measurements across centers was 3.1% (95% CI: [2.9%, 3.2%]), demonstrating the high precision of 454 sequencing to detect mutations. Additionally, we took advantage of the high sensitivity of deep-sequencing. As such, we observed 7 distinct novel mutations (n=2 TET2, n=3 CBL, n=2 KRAS) with frequencies below the Sanger sequencing cut-off value of 20% (median values ranging from 2.8% - 12.6%). These low-level mutations were consistently detected in all laboratories (one CBL mutation with <3% frequency detected in only 5/8 centers). In conclusion, we here demonstrate in a multicenter analysis that amplicon-based deep-sequencing is technically feasible, achieves a high concordance across multiple laboratories, and therefore allows a broad and in-depth molecular characterization of hematological malignancies with high diagnostic sensitivity. Disclosures: Kohlmann: MLL Munich Leukemia Laboratory: Employment. Garicochea:Roche Diagnostics: Research Funding. Grossmann:MLL Munich Leukemia Laboratory: Employment. Hanczaruk:454 Life Sciences: Employment. Jansen:Roche Diagnostics: Research Funding. te Kronnie:Roche Diagnostics: Research Funding. Martinelli:Roche Diagnostics: Research Funding. McGowan:454 Life Sciences: Employment. Stabentheiner:Roche Diagnostics: Research Funding. Timmermann:Roche Diagnostics: Research Funding. Vandenberghe:Roche Diagnostics: Research Funding. Young:Roche Diagnostics: Research Funding. Dugas:Roche Diagnostics: Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership; Roche Diagnostics: Research Funding.
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Unselt, Desiree, Katherine Knudsen, Christopher Rounds, Janet Doolittle-Hall, Fernando Torres, Jennifer Sims, and Jennifer Mason. "Abstract 437: Characterization of SARS-CoV-2 using the Ion AmpliSeq SARS-CoV-2 research panel." Cancer Research 82, no. 12_Supplement (June 15, 2022): 437. http://dx.doi.org/10.1158/1538-7445.am2022-437.
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Abstract Background: The rapid spread of COVID-19 has resulted in an urgent need for effective diagnostic and therapeutic strategies against SARS-CoV-2. Next-generation sequencing (NGS) is a powerful tool in the identification and characterization of this pathogen and genomic information may aid in understanding the mechanisms of therapeutic resistance, vaccine escape, virulence, and pathogenicity. The Ion AmpliSeq SARS-CoV-2 Research Panel is a targeted NGS solution that facilitates sequence analysis of the SARS-CoV-2 genome. Paired with a bioinformatics assembly and variant calling pipelines, this assay allows for accurate characterization of the dominant SARS-CoV-2 variant. This assay’s performance was analytically validated for the detection of mutations (substitutions, insertions, and deletions) in RNA derived from nasopharyngeal (NP) swabs. Method: The Ion AmpliSeq SARS-CoV-2 Research panel consists of two primer pair pools generating 237 amplicons specific to the SARS-CoV-2 virus. Reverse transcription of the RNA was performed using the SuperScript VILO cDNA Synthesis kit. Library preparation was then completed using the Ion AmpliSeq Library Kit Plus kit. The final library was quantified, normalized, pooled, and sequenced. Raw sequencing data was aligned to the AmpliSeq SARS-CoV-2 Research panel, using the MN908947.3 reference genome. Variants were called using the Torrent Variant Caller and annotated using the COVID19AnnotateSnpEff plugin. The reference-guided iterative assembler IRMA was used to produce a single consensus sequence consisting of the reference genome sequence modified to include sequence variations supported by the reads. The Pangolin COVID-19 lineage assigner software tool was used to assign SARS-CoV-2 lineage. Analytical validation was completed using controls (Twist Biosciences, BEI Resources, ATCC) and RNA derived from NP swabs. Accuracy and specificity were examined by evaluating the correctness of calling true negative variants compared to false positive and all other variant calls, respectively. Precision and limit of detection (LoD) were examined by evaluating the concordance of variants across replicate samples. Limit of Blank (LoB) was calculated as the 95th percentile of reads per amplicon in the negative samples. Results: Accuracy of base calling, specificity, and precision were 100% for SNVs, insertions, and deletions above 25% allele frequency. LoD was determined to be 576 viral copies/mL. LoB was determined to be 202 reads per amplicon. Pangolin lineage assignment was 100% for all samples. Conclusions: This panel accurately characterizes SARS-CoV-2 variants, allowing for accurate consensus sequence generation, mutation annotation, and lineage assignment. Citation Format: Desiree Unselt, Katherine Knudsen, Christopher Rounds, Janet Doolittle-Hall, Fernando Torres, Jennifer Sims, Jennifer Mason. Characterization of SARS-CoV-2 using the Ion AmpliSeq SARS-CoV-2 research panel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 437.
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Glenn, Travis C., Todd W. Pierson, Natalia J. Bayona-Vásquez, Troy J. Kieran, Sandra L. Hoffberg, Jesse C. Thomas IV, Daniel E. Lefever, et al. "Adapterama II: universal amplicon sequencing on Illumina platforms (TaggiMatrix)." PeerJ 7 (October 11, 2019): e7786. http://dx.doi.org/10.7717/peerj.7786.
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Next-generation sequencing (NGS) of amplicons is used in a wide variety of contexts. In many cases, NGS amplicon sequencing remains overly expensive and inflexible, with library preparation strategies relying upon the fusion of locus-specific primers to full-length adapter sequences with a single identifying sequence or ligating adapters onto PCR products. In Adapterama I, we presented universal stubs and primers to produce thousands of unique index combinations and a modifiable system for incorporating them into Illumina libraries. Here, we describe multiple ways to use the Adapterama system and other approaches for amplicon sequencing on Illumina instruments. In the variant we use most frequently for large-scale projects, we fuse partial adapter sequences (TruSeq or Nextera) onto the 5′ end of locus-specific PCR primers with variable-length tag sequences between the adapter and locus-specific sequences. These fusion primers can be used combinatorially to amplify samples within a 96-well plate (8 forward primers + 12 reverse primers yield 8 × 12 = 96 combinations), and the resulting amplicons can be pooled. The initial PCR products then serve as template for a second round of PCR with dual-indexed iTru or iNext primers (also used combinatorially) to make full-length libraries. The resulting quadruple-indexed amplicons have diversity at most base positions and can be pooled with any standard Illumina library for sequencing. The number of sequencing reads from the amplicon pools can be adjusted, facilitating deep sequencing when required or reducing sequencing costs per sample to an economically trivial amount when deep coverage is not needed. We demonstrate the utility and versatility of our approaches with results from six projects using different implementations of our protocols. Thus, we show that these methods facilitate amplicon library construction for Illumina instruments at reduced cost with increased flexibility. A simple web page to design fusion primers compatible with iTru primers is available at: http://baddna.uga.edu/tools-taggi.html. A fast and easy to use program to demultiplex amplicon pools with internal indexes is available at: https://github.com/lefeverde/Mr_Demuxy.
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Delavaux, Camille S., Robert J. Ramos, Sidney L. Sturmer, and James D. Bever. "Environmental identification of arbuscular mycorrhizal fungi using the LSU rDNA gene region: an expanded database and improved pipeline." Mycorrhiza 32, no. 2 (January 31, 2022): 145–53. http://dx.doi.org/10.1007/s00572-022-01068-3.
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AbstractArbuscular mycorrhizal fungi (AMF; Glomeromycota) are difficult to culture; therefore, establishing a robust amplicon-based approach to taxa identification is imperative to describe AMF diversity. Further, due to low and biased sampling of AMF taxa, molecular databases do not represent the breadth of AMF diversity, making database matching approaches suboptimal. Therefore, a full description of AMF diversity requires a tool to determine sequence-based placement in the Glomeromycota clade. Nonetheless, commonly used gene regions, including the SSU and ITS, do not enable reliable phylogenetic placement. Here, we present an improved database and pipeline for the phylogenetic determination of AMF using amplicons from the large subunit (LSU) rRNA gene. We improve our database and backbone tree by including additional outgroup sequences. We also improve an existing bioinformatics pipeline by aligning forward and reverse reads separately, using a universal alignment for all tree building, and implementing a BLAST screening prior to tree building to remove non-homologous sequences. Finally, we present a script to extract AMF belonging to 11 major families as well as an amplicon sequencing variant (ASV) version of our pipeline. We test the utility of the pipeline by testing the placement of known AMF, known non-AMF, and Acaulospora sp. spore sequences. This work represents the most comprehensive database and pipeline for phylogenetic placement of AMF LSU amplicon sequences within the Glomeromycota clade.
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Lipsky, Robert H., Chiara M. Mazzanti, Joseph G. Rudolph, Ke Xu, Gopal Vyas, David Bozak, Marta Q. Radel, and David Goldman. "DNA Melting Analysis for Detection of Single Nucleotide Polymorphisms." Clinical Chemistry 47, no. 4 (April 1, 2001): 635–44. http://dx.doi.org/10.1093/clinchem/47.4.635.
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Abstract Background: Several methods for detection of single nucleotide polymorphisms (SNPs; e.g., denaturing gradient gel electrophoresis and denaturing HPLC) are indirectly based on the principle of differential melting of heteroduplex DNA. We present a method for detecting SNPs that is directly based on this principle. Methods: We used a double-stranded DNA-specific fluorescent dye, SYBR Green I (SYBR) in an efficient system (PE 7700 Sequence Detector) in which DNA melting was controlled and monitored in a 96-well plate format. We measured the decrease in fluorescence intensity that accompanied DNA duplex denaturation, evaluating the effects of fragment length, dye concentration, DNA concentration, and sequence context using four naturally occurring polymorphisms (three SNPs and a single-base deletion/insertion). Results: DNA melting analysis (DM) was used successfully for variant detection, and we also discovered two previously unknown SNPs by this approach. Concentrations of DNA amplicons were readily monitored by SYBR fluorescence, and DNA amplicon concentrations were highly reproducible, with a CV of 2.6%. We readily detected differences in the melting temperature between homoduplex and heteroduplex fragments 15–167 bp in length and differing by only a single nucleotide substitution. Conclusions: The efficiency and sensitivity of DMA make it highly suitable for the large-scale detection of sequence variants.
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Montgomery, Jesse, Carl T. Wittwer, Jana O. Kent, and Luming Zhou. "Scanning the Cystic Fibrosis Transmembrane Conductance Regulator Gene Using High-Resolution DNA Melting Analysis." Clinical Chemistry 53, no. 11 (November 1, 2007): 1891–98. http://dx.doi.org/10.1373/clinchem.2007.092361.
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Abstract Background: Complete gene analysis of the cystic fibrosis transmembrane conductance regulator gene (CFTR) by scanning and/or sequencing is seldom performed because of the cost, time, and labor involved. High-resolution DNA melting analysis is a rapid, closed-tube alternative for gene scanning and genotyping. Methods: The 27 exons of CFTR were amplified in 37 PCR products under identical conditions. Common variants in 96 blood donors were identified in each exon by high-resolution melting on a LightScanner®. We then performed a subsequent blinded study on 30 samples enriched for disease-causing variants, including all 23 variants recommended by the American College of Medical Genetics and 8 additional, well-characterized variants. Results: We identified 22 different sequence variants in 96 blood donors, including 4 novel variants and the disease-causing p.F508del. In the blinded study, all 40 disease-causing heterozygotes (29 unique) were detected, including 1 new probable disease-causing variant (c.3500-2A>T). The number of false-positive amplicons was decreased 96% by considering the 6 most common heterozygotes. The melting patterns of most heterozygotes were unique (37 of 40 pairs within the same amplicon), the exceptions being p.F508del vs p.I507del, p.G551D vs p.R553X, and p.W1282X vs c.4002A>G. The homozygotes p.G542X, c.2789 + 5G>A, and c.3849 + 10kbC>T were directly identified, but homozygous p.F508del was not. Specific genotyping of these exceptions, as well as genotyping of the 5T allele of intron 8, was achieved by unlabeled-probe and small-amplicon melting assays. Conclusions: High-resolution DNA melting methods provide a rapid and accurate alternative for complete CFTR analysis. False positives can be decreased by considering the melting profiles of common variants.
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Liu, Po‐Yu, Shan‐Hua Yang, and Sung‐Yin Yang. "KTU: K‐mer Taxonomic Units improve the biological relevance of amplicon sequence variant microbiota data." Methods in Ecology and Evolution 13, no. 3 (November 12, 2021): 560–68. http://dx.doi.org/10.1111/2041-210x.13758.
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Gavriliuc, Stefan, Mason R. Stothart, Astrid Henry, and Jocelyn Poissant. "Long-term storage of feces at −80 °C versus −20 °C is negligible for 16S rRNA amplicon profiling of the equine bacterial microbiome." PeerJ 9 (March 9, 2021): e10837. http://dx.doi.org/10.7717/peerj.10837.
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The development of next-generation sequencing technologies has spurred a surge of research on bacterial microbiome diversity and function. But despite the rapid growth of the field, many uncertainties remain regarding the impact of differing methodologies on downstream results. Sample storage temperature is conventionally thought to be among the most important factors for ensuring reproducibility across marker gene studies, but to date much of the research on this topic has focused on short-term storage in the context of clinical applications. Consequently, it has remained unclear if storage at −80 °C, widely viewed as the gold standard for long-term archival of feces, is truly required for maintaining sample integrity in amplicon-based studies. A better understanding of the impacts of long-term storage conditions is important given the substantial cost and limited availability of ultra-low temperature freezers. To this end, we compared bacterial microbiome profiles inferred from 16S V3–V4 amplicon sequencing for paired fecal samples obtained from a feral horse population from Sable Island, Nova Scotia, Canada, stored at either −80 °C or −20 °C for 4 years. We found that storage temperature did not significantly affect alpha diversity measures, including amplicon sequence variant (ASV) richness and evenness, and abundance of rare sequence variants, nor presence/absence, relative abundances and phylogenetic diversity weighted measures of beta diversity. These results indicate that storage of equine feces at −20 °C for periods ranging from a few months to a few years is equivalent to storage at −80 °C for amplicon-based microbiome studies, adding to accumulating evidence indicating that standard domestic freezers are both economical and effective for microbiome research.
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Murley, Alexander G., Yu Nie, Zoe Golder, Michael John Keogh, Colin Smith, James W. Ironside, and Patrick F. Chinnery. "High-Depth PRNP Sequencing in Brains With Sporadic Creutzfeldt-Jakob Disease." Neurology Genetics 9, no. 1 (January 19, 2023): e200054. http://dx.doi.org/10.1212/nxg.0000000000200054.
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Background and ObjectivesSporadic Creutzfeldt-Jakob disease (sCJD) has established genetic risk factors, but, in contrast to genetic and acquired CJD, the initial trigger for misfolded prion aggregation and spread is not known. In this study, we tested the hypotheses that pathologic somatic variants in the prion genePRNPare increased in sCJD, potentially leading to the seeding of misfolded prion protein.MethodsHigh-depth amplicon-based short read sequencing of thePRNPcoding region was performed on postmortem brain tissue from patients with a clinical and neuropathologic diagnosis of sCJD (n = 142), Alzheimer disease (AD) (n = 51) and controls with no clinical or neuropathologic diagnosis of a neurodegenerative disease (n = 71). Each DNA sample was sequenced twice, including independent PCR amplification, library preparation, and sequencing. We used RePlow to call somatic variants with high sensitivity and specificity and optimal sequence kernel association test to compare variant burden between groups.ResultsTwo sCJD cases had somatic (variant allele frequency 0.5–1%)PRNPvariants not previously identified, but with high in silico predicated pathogenicity. However, the pathogenicity of these variants is uncertain, as both located in the octapeptide repeat region where no point variations have previously been associated with sCJD. There was no overall difference in burden somaticPRNPin sCJD compared with controls and a lower burden compared with Alzheimer disease.DiscussionSomatic variants inPRNPare unlikely to play a major role in sCJD but may contribute to the disease mechanism in a minority of cases.
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Faleye, Temitope O. C., Erin Driver, Devin Bowes, Sangeet Adhikari, Deborah Adams, Arvind Varsani, Rolf U. Halden, and Matthew Scotch. "Pan-Enterovirus Amplicon-Based High-Throughput Sequencing Detects the Complete Capsid of a EVA71 Genotype C1 Variant via Wastewater-Based Epidemiology in Arizona." Viruses 13, no. 1 (January 7, 2021): 74. http://dx.doi.org/10.3390/v13010074.
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We describe the complete capsid of a genotype C1-like Enterovirus A71 variant recovered from wastewater in a neighborhood in the greater Tempe, Arizona area (Southwest United States) in May 2020 using a pan-enterovirus amplicon-based high-throughput sequencing strategy. The variant seems to have been circulating for over two years, but its sequence has not been documented in that period. As the SARS-CoV-2 pandemic has resulted in changes in health-seeking behavior and overwhelmed pathogen diagnostics, our findings highlight the importance of wastewater-based epidemiology (WBE ) as an early warning system for virus surveillance.
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Faleye, Temitope O. C., Erin Driver, Devin Bowes, Sangeet Adhikari, Deborah Adams, Arvind Varsani, Rolf U. Halden, and Matthew Scotch. "Pan-Enterovirus Amplicon-Based High-Throughput Sequencing Detects the Complete Capsid of a EVA71 Genotype C1 Variant via Wastewater-Based Epidemiology in Arizona." Viruses 13, no. 1 (January 7, 2021): 74. http://dx.doi.org/10.3390/v13010074.
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We describe the complete capsid of a genotype C1-like Enterovirus A71 variant recovered from wastewater in a neighborhood in the greater Tempe, Arizona area (Southwest United States) in May 2020 using a pan-enterovirus amplicon-based high-throughput sequencing strategy. The variant seems to have been circulating for over two years, but its sequence has not been documented in that period. As the SARS-CoV-2 pandemic has resulted in changes in health-seeking behavior and overwhelmed pathogen diagnostics, our findings highlight the importance of wastewater-based epidemiology (WBE ) as an early warning system for virus surveillance.
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Rueca, Martina, Emanuela Giombini, Francesco Messina, Barbara Bartolini, Antonino Di Caro, Maria Rosaria Capobianchi, and Cesare EM Gruber. "The Easy-to-Use SARS-CoV-2 Assembler for Genome Sequencing: Development Study." JMIR Bioinformatics and Biotechnology 3, no. 1 (March 14, 2022): e31536. http://dx.doi.org/10.2196/31536.
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Background Early sequencing and quick analysis of the SARS-CoV-2 genome have contributed to the understanding of the dynamics of COVID-19 epidemics and in designing countermeasures at a global level. Objective Amplicon-based next-generation sequencing (NGS) methods are widely used to sequence the SARS-CoV-2 genome and to identify novel variants that are emerging in rapid succession as well as harboring multiple deletions and amino acid–changing mutations. Methods To facilitate the analysis of NGS sequencing data obtained from amplicon-based sequencing methods, here, we propose an easy-to-use SARS-CoV-2 genome assembler: the Easy-to-use SARS-CoV-2 Assembler (ESCA) pipeline. Results Our results have shown that ESCA could perform high-quality genome assembly from Ion Torrent and Illumina raw data and help the user in easily correct low-coverage regions. Moreover, ESCA includes the possibility of comparing assembled genomes of multisample runs through an easy table format. Conclusions In conclusion, ESCA automatically furnished a variant table output file, fundamental to rapidly recognizing variants of interest. Our pipeline could be a useful method for obtaining a complete, rapid, and accurate analysis even with minimal knowledge in bioinformatics.
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Del Prete, Gregory Q., Haesun Park, Christine M. Fennessey, Carolyn Reid, Leslie Lipkey, Laura Newman, Kelli Oswald, et al. "Molecularly Tagged Simian Immunodeficiency Virus SIVmac239 Synthetic Swarm for Tracking Independent Infection Events." Journal of Virology 88, no. 14 (May 7, 2014): 8077–90. http://dx.doi.org/10.1128/jvi.01026-14.
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ABSTRACTFollowing mucosal human immunodeficiency virus type 1 transmission, systemic infection is established by one or only a few viral variants. Modeling single-variant, mucosal transmission in nonhuman primates using limiting-dose inoculations with a diverse simian immunodeficiency virus isolate stock may increase variability between animals since individual variants within the stock may have substantial functional differences. To decrease variability between animals while retaining the ability to enumerate transmitted/founder variants by sequence analysis, we modified the SIVmac239 clone to generate 10 unique clones that differ by two or three synonymous mutations (molecular tags). Transfection- and infection-derived virus stocks containing all 10 variants showed limited phenotypic differences in 9 of the 10 clones. Twenty-nine rhesus macaques were challenged intrarectally or intravenously with either a single dose or repeated, limiting doses of either stock. The proportion of each variant within each inoculum and in plasma from infected animals was determined by using a novel real-time single-genome amplification assay. Each animal was infected with one to five variants, the number correlating with the dose. Longitudinal sequence analysis revealed that the molecular tags are highly stable with no reversion to the parental sequence detected in >2 years of follow-up. Overall, the viral stocks are functional and mucosally transmissible and the number of variants is conveniently discernible by sequence analysis of a small amplicon. This approach should be useful for tracking individual infection events in preclinical vaccine evaluations, long-term viral reservoir establishment/clearance research, and transmission/early-event studies.IMPORTANCEHuman immunodeficiency virus type 1 transmission is established by one or only a few viral variants. Modeling of limited variant transmission in nonhuman primates with a diverse simian immunodeficiency virus isolate stock may increase the variability between animals because of functional differences in the individual variants within the stock. To decrease such variability while retaining the ability to distinguish and enumerate transmitted/founder variants by sequence analysis, we generated a viral stock with 10 sequence-identifiable but otherwise genetically identical variants. This virus was characterizedin vitroandin vivoand shown to allow discrimination of distinct transmission events. This approach provides a novel nonhuman primate challenge system for the study of viral transmission, evaluation of vaccines and other prevention approaches, and characterization of viral reservoirs and strategies to target them.
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Harbuzov, Zoya, Valeria Farberova, Moshe Tom, Alberto Pallavicini, David Stanković, Tamar Lotan, and Hadas Lubinevsky. "Amplicon sequence variant-based meiofaunal community composition revealed by DADA2 tool is compatible with species composition." Marine Genomics 65 (October 2022): 100980. http://dx.doi.org/10.1016/j.margen.2022.100980.
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Seipp, Michael T., David Pattison, Jacob D. Durtschi, Mohamed Jama, Karl V. Voelkerding, and Carl T. Wittwer. "Quadruplex Genotyping of F5, F2, and MTHFR Variants in a Single Closed Tube by High-Resolution Amplicon Melting." Clinical Chemistry 54, no. 1 (January 1, 2008): 108–15. http://dx.doi.org/10.1373/clinchem.2007.097121.
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Abstract Background: Multiplexed amplicon melting is a closed-tube method for genotyping that does not require probes, real-time analysis, asymmetric PCR, or allele-specific PCR; however, correct differentiation of homozygous mutant and wild-type samples by melting temperature (Tm) analysis requires high-resolution melting analysis and controlled reaction conditions. Methods: We designed 4 amplicons bracketing the F5 [coagulation factor V (proaccelerin, labile factor)] 1691G>A, MTHFR (NADPH) 1298A>C, MTHFR 677C>T, and F2 [coagulation factor II (thrombin)] 20210G>A gene variants to melt at different temperatures by varying amplicon length and adding GC- or AT-rich 5′ tails to selected primers. We used rapid-cycle PCRs with cycles of 19–23 s in the presence of a saturating DNA dye and temperature-correction controls and then conducted a high-resolution melting analysis. Heterozygotes were identified at each locus by curve shape, and homozygous genotypes were assigned by Tm. We blinded samples previously genotyped by other methods before analysis with the multiplex melting assay (n = 110). Results: All samples were correctly genotyped with the exception of 7 MTHFR 1298 samples with atypical melting profiles that could not be assigned. Sequencing revealed that these 5 heterozygotes and 2 homozygotes contained the unexpected sequence variant MTHFR 1317T>C. The use of temperature-correction controls decreased the Tm SD within homozygotes by a mean of 38%. Conclusion: Rapid-cycle PCR with high-resolution melting analysis allows simple and accurate multiplex genotyping to at least a factor of 4.
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DINESH, KRISHANENDER, ARCHANA VERMA, I. D. GUPTA, and S. K. DASH. "Association of polymorphic variant of exons 6 and 11 of lactoferrin gene with mastitis in Murrah buffalo." Indian Journal of Animal Sciences 90, no. 4 (September 1, 2020): 588–91. http://dx.doi.org/10.56093/ijans.v90i4.104206.
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Lactoferrin is one of the important candidate genes for mastitis resistance in dairy animals. The gene is located on Bos taurus autosome (BTA) 22 and consists of 17 exons spanning over 34.5 kb of genomic DNA. The present study was undertaken to identify allelic variant in exons 6 and 11 of lactoferrin gene and to analyze association with incidence of clinical mastitis in Murrah buffalo. The amplification of exons 6 and 11 of lactoferrin gene yielded 301 and 131 bp amplicon size. Comparison of nucleotide sequence of exonic region of lactoferrin gene with Bos taurus (NCBI accession number AC_000179.1) revealed 6 mutations; among them 3 were in coding DNA sequence and remaining 3 were in flanking intronic region. All these mutations were found in exon 6 and synonymous in nature without affecting the sequence of amino acid. PCR-restriction fragment length polymorphism (RFLP) analysis of 301 bp amplicon using FokI restriction enzyme exhibited polymorphic pattern with two genotypes (AA and AB) with respective frequency of 0.625 and 0.375. The frequencies of two alleles, A and B were estimated as 0.81 and 0.19 respectively. RE Hpy188I and HinfI used for digestion of exon 11 had exhibited monomorphic pattern. The chi-square (χ2) analysis revealed significant association between incidence of clinical mastitis and genetic variant of exon 6 and animals with AA genotype were found to be less susceptible to mastitis. The findings indicate potential scope for incorporation of lactoferrin gene in selection and breeding of Murrah buffaloes for improved genetic resistance to mastitis.
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Looney, Timothy, Alexander Glavin, Sarabjot Pabla, Sean T. Glenn, Lauren Miller, Denise Topacio-Hall, Elizabeth Linch, et al. "Long-amplicon TCRβ repertoire sequencing to reveal human T-cell receptor variable gene polymorphism: Implications for the prediction and interpretation of immunotherapy outcome." Journal of Clinical Oncology 36, no. 5_suppl (February 10, 2018): 129. http://dx.doi.org/10.1200/jco.2018.36.5_suppl.129.
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129 Background: Human T cell antigen receptors play a critical role in protective immune responses but are also implicated in autoimmune disease and immune-mediated adverse events during immunotherapy. The antigen specificity of the T cell receptor is determined in part by the sequence of the CDR and Framework regions encoded by the TCRB variable gene. Previous studies of population sequencing data indicate that current antigen receptor allele databases, such as IMGT, fail to capture a significant portion of human variation. Here we use long-amplicon multiplex sequencing of rearranged TCRB receptors to validate putative novel human variable gene alleles previously recovered from 1000 genomes data. Methods: TCRB rearrangements were amplified from cDNA from 85 Caucasians undergoing treatment for melanoma using AmpliSeq-based multiplex Framework 1 and Constant gene primers to produce ~330bp amplicons. Samples were sequenced using the Ion Torrent S5 530 chip to produce ~1.5M raw reads per sample. Ion Reporter was used for clonotyping and identification of variable gene sequences absent from the IMGT database. Putatively novel sequences were compared to those reported in the Lym1k database of alleles recovered from 1000 genomes data. Results: We identified 15 novel variable gene alleles that are absent from the IMGT database and result in amino acid changes to the CDR or Framework regions of the TCR. Typically, a single individual was found to be heterozygous for a novel variant, though we note two instances where multiple individuals possessed a novel variant. We also identified novel variable gene alleles that are absent from the Lym1k database, potentially due to challenges in inferring receptor alleles from short-read population sequencing studies. Conclusions: We find evidence for significant human diversity in TCRB variable gene alleles beyond what is currently represented in the IMGT database. TCRB sequencing using multiplex Framework 1 and Constant gene targeting primers is ideally suited for studying the role of TCRB polymorphism in autoimmune disease and immune-mediated adverse events during immunotherapy.
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Campoccia, Davide, Lucio Montanaro, Stefano Ravaioli, Ilaria Cangini, Pietro Speziale, and Carla Renata Arciola. "Description of a New Group of Variants of the Staphylococcus Aureus Elastin-Binding Protein that Lacks an Entire DNA Segment of 180 bp." International Journal of Artificial Organs 32, no. 9 (September 2009): 621–29. http://dx.doi.org/10.1177/039139880903200911.
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The elastin-binding protein (EbpS) is a microbial surface component recognizing adhesive matrix molecule (MSCRAMM) found in Staphylococcus aureus that mediates bacterial cell binding to soluble elastin and tropoelastin. In scientific literature it is well established that the gene encoding for the elastin-binding protein (ebpS) is present in the vast majority of Staphylococcus aureus clinical isolates. The present study aimed at investigating a group of new variant forms of ebpS gene identified in S. aureus clinical strains isolated from implant-related orthopedic infections. A PCR screening for the ebpS gene, conducted on over two hundred S. aureus clinical isolates from implant-related infections revealed the detection of six strains exhibiting an altered amplicon size, shorter than expected. In order to elucidate the sequence changes present in these gene variants, the trait comprised between the primers was analyzed in all six isolates bearing the modification and in four isolates exhibiting the regular amplicon size. A similar form of the ebpS gene, lacking a DNA trait of 180 bp, was confirmed in all six isolates independently of their clonal origin. Interestingly, only three of these isolates, all with type IV polymorphism of the accessory genes regulator (agr) locus, showed exactly the same sequence and, thus, the same pattern of point mutations with respect to reference strains. From nucleotide translation, the corresponding encoded protein was found to lack an entire peptide segment of 60 amino acids. From nucleotide sequence translation, this modification was found to implicate the disappearance of an entire hydrophobic domain, whose functional significance needs to be further investigated.
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Rivers, Adam R., Kyle C. Weber, Terrence G. Gardner, Shuang Liu, and Shalamar D. Armstrong. "ITSxpress: Software to rapidly trim internally transcribed spacer sequences with quality scores for marker gene analysis." F1000Research 7 (September 6, 2018): 1418. http://dx.doi.org/10.12688/f1000research.15704.1.
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The internally transcribed spacer (ITS) region between the small subunit ribosomal RNA gene and large subunit ribosomal RNA gene is a widely used phylogenetic marker for fungi and other taxa. The eukaryotic ITS contains the conserved 5.8S rRNA and is divided into the ITS1 and ITS2 hypervariable regions. These regions are variable in length and are amplified using primers complementary to the conserved regions of their flanking genes. Previous work has shown that removing the conserved regions results in more accurate taxonomic classification. An existing software program, ITSx, is capable of trimming FASTA sequences by matching hidden Markov model profiles to the ends of the conserved genes using the software suite HMMER. ITSxpress was developed to extend this technique from marker gene studies using Operational Taxonomic Units (OTU’s) to studies using exact sequence variants; a method used by the software packages Dada2, Deblur, QIIME 2, and Unoise. The sequence variant approach uses the quality scores of each read to identify sequences that are statistically likely to represent real sequences. ITSxpress enables this by processing FASTQ rather than FASTA files. The software also speeds up the trimming of reads by a factor of 14-23 times on a 4-core computer by temporarily clustering highly similar sequences that are common in amplicon data and utilizing optimized parameters for Hmmsearch. ITSxpress is available as a QIIME 2 plugin and a stand-alone application installable from the Python package index, Bioconda, and Github.
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He, Yuqing, Francesco Tiezzi, Jeremy T. Howard, Yijian Huang, Kent A. Gray, and Christian Maltecca. "23 Exploring the Role of Gut Microbiota in Host Feeding Behavior Among Breeds in Swine." Journal of Animal Science 99, Supplement_3 (October 8, 2021): 10–11. http://dx.doi.org/10.1093/jas/skab235.019.
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Abstract The present study aimed to explore gut microbiota’s overall influence on feeding behavior and identify specific microbes associated with the traits in three commercial swine breeds at different growth stages. Feeding behavior measures were obtained from 651 pigs of three breeds (Duroc, Landrace, and Large White) from 67 to 175 days of age. Seven feeding behavior traits cover the information of feed intake, feeder occupation time, feeding rate, and the number of visits to the feeder. Rectal swabs were collected from each pig at 73 ± 3, 123 ± 4, and 158 ± 4 days of age. DNA was extracted and subjected to 16S rRNA gene sequencing. Sequences were analyzed using the QIIME2. The amplicon sequence variant table was constructed using the DADA2. Mixed linear models were used to perform statistical analyses. Differences in feeding behavior traits among breeds and across time periods were found. The proportion of phenotypic variances explained by the microbiome ranged from 0.10 to 0.26, 0.14 to 0.25, and 0.12 to 0.28 in Duroc pigs, from 0.09 to 0.20, 0.10 to 0.23, and 0.12 to 0.23 in Landrace pigs, from 0.10 to 0.17, 0.11 to 0.19, and 0.10 to 0.31 in Large White pigs at three time points, respectively. A total of 21, 10, and 35 amplicon sequence variants were found to be significantly (q-value < 0.05) associated with feeding behavior traits for Duroc, Landrace, and Large White across three time points. This study demonstrated the importance of the gut microbiome composition in influencing the host feeding behavior and identified multiple microbes associated with the feeding behavior of pigs from either Duroc, Landrace, or Large White breeds at three growth stages. Our study provides insight into the interaction between gut microbiota and feeding behavior and highlights the breed effects in swine microbial studies.
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Bernardy, M. G., C. J. French, M. Milks, and G. Jesperson. "New Variant of Little cherry virus Associated with Little Cherry Disease of Sweet Cherry in British Columbia, Canada." Plant Disease 86, no. 12 (December 2002): 1406. http://dx.doi.org/10.1094/pdis.2002.86.12.1406c.
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Little cherry disease (LChD) occurs in most cherry growing areas in the world. Infection of sensitive cultivars results in small fruit with poor color, angular shape, and insipid flavor. Three viruses associated with LCD have been described: (i) Little cherry virus-1 (LChV-1) first found and described in Germany (4); (ii) LChV-2 an isolate obtained from the United States (2); and (iii) LChV-3 first found and described in British Columbia (1). Despite similarities in symptom development in orchard trees and woody indexing, the three viruses have distinct molecular sequences (3). LChV-2 and -3 share greater homology with each other than either does with LChV-1. For many years, the British Columbia Ministry of Agriculture, Fisheries and Food (BCMAFF) in conjunction with Agriculture and Agri-Food Canada, Pacific Agri-Food Research Centre (AAFC-PARC) has conducted a survey to monitor the incidence and spread of LChD in the Okanagan and Kootenay valleys of British Columbia, Canada. Until recently, testing for LChD used woody indexing on indicator trees, Prunus avium cv. Lambert (fruit symptoms) and cvs. Canindex1 and Sam (foliar symptoms). Recently, incidence of LChD has been evaluated using double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) developed at AAFC-PARC (3), and reverse transcription-polymerase chain reaction (RT-PCR) tests based on sequence data from LChV-3 (1). During the 1999 survey, orchard trees displaying symptoms typical of LChD tested negative for LChV-3 using ELISA and RT-PCR. Also, trees that formerly tested positive for LChD by woody indexing also tested negative for LChV-3 using RT-PCR and ELISA. Two hundred ninety-three trees were subsequently tested for LChV-1 by RT-PCR using the primer set LCV3EC/LCV16659 (4). A 276-bp fragment corresponding to the extreme 3′ untranslated region (3′ UTR) of the LChV-1 genome was amplified by RT-PCR from 140 of the trees. The RT-PCR amplicon from one sample (#99-68B from Peachland) was sequenced and using a BLASTn search, LChV-1 was identified as the most probable match (E value 4e-59). Sequence alignment using ClustalX identified two regions of high sequence homology; bases 2 to 70 (92%) and bases 99 to 239 (95%). The intervening region displayed much lower homology (61%). The overall homology of the amplicon was 88% compared to the corresponding region in LChV-1. Divergence between the published sequence of LChV-1 and the sequence of the new LChV isolate (tentatively named LChV-4) was investigated. Seven sets of primers constructed on the basis of sequence data from various regions of the LChV-1 genome (two sets from each of the RNA-dependent RNA polymerase, heat shock 70 protein homologue, and coat protein) failed to yield RT-PCR products when tested with the LChV-4 isolate. LChV-4 is clearly related to LChV-1 within the 3′-UTR but complete sequencing is required to determine the overall relationship with other viruses causing LChD. The discovery of a new isolate of LChV in British Columbia may require a reevaluation of the epidemiology of LChD and disease management strategy. References: (1) K. C. Eastwell and M. G. Bernardy. Phytopathology 91:268, 2001. (2) M. E. Rott, and W. Jelkmann. Phytopathology 91:261, 2001. (3) J. Theilmann et al. Phytopathology 92:87, 2002. (4) M. Vitushkina et al. Eur. J. Plant Pathol. 103;803, 1997.
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Claus, Marlise P., Michele Lunardi, Alice F. Alfieri, Daniele Sartori, Maria Helena P. Fungaro, and Amauri A. Alfieri. "Identification of the recently described new type of bovine papillomavirus (BPV-8) in a Brazilian beef cattle herd." Pesquisa Veterinária Brasileira 29, no. 1 (January 2009): 25–28. http://dx.doi.org/10.1590/s0100-736x2009000100003.
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Bovine papillomavirus type 8 (BPV-8) was first detected and described in teat warts as well as in healthy teat skin from cattle raised in Japan. The entire viral genome was sequenced in 2007. Additionally, a variant of BPV-8, BPV-8-EB, was also identified from papillomatous lesions of a European bison in Slovakia. In Brazil, despite the relatively common occurrence of BPV infections, the identification and determination of viral types present in cattle is still sporadic. The aim of this study is to report the occurrence of the recently described BPV-8 in Brazil. The virus was identified in a skin warts obtained from a beef cattle herd located in Parana state, southern Brazil. The papilloma had a macular, non-verrucous gross aspect and was located on the dorsal thorax of a cow. Polymerase chain reaction (PCR) was performed using generic primers for partial amplification of L1 gene. The obtained amplicon (480bp) was cloned and two selected clones were sequenced. The nucleotide sequence was compared to existing papillomaviral genomic sequences, identifying the virus as BPV type 8. This study represents the first report of BPV-8 occurrence in Brazil, what suggests its presence among Brazilian cattle.
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Yen, Sandi, Jethro Johnson, and Nicholas E. Ilott. "Streamlined processing and analysis of 16S rRNA amplicon sequencing data with OCMS_16S and OCMSlooksy." Wellcome Open Research 7 (February 23, 2022): 68. http://dx.doi.org/10.12688/wellcomeopenres.17632.1.
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16S rRNA gene sequencing is a cost-effective method for profiling the bacterial component of a microbiome. Nevertheless, processing and analysis of the resulting sequencing data is often constrained by the availability of dedicated bioinformaticians - creating a bottleneck for biological interpretation. Multiple visualisation and analysis tools now exist for downstream analysis of 16S rRNA data. These tools are designed with biological scientists in mind and therefore consist of a graphical user interface that interacts with taxonomic counts tables to perform tasks such as alpha- and beta-diversity analysis and differential abundance. However, generating the input to these applications still relies on bioinformatics experience, creating a disconnect between data processing and data analysis. We aimed to bridge the gap between data processing and data analysis. To do this we have created two tools - OCMS_16S and OCMSlooksy - that perform data processing and data visualisation/analysis, respectively. OCMS_16S is a cgat-core based pipeline that wraps DADA2 functionality in order to facilitate processing of raw sequence reads into tables of amplicon sequence variant (ASV) counts using a simple command line interface. OCMSlooksy is an RShiny application that takes an OCMS_16S-generated SQLite database as input to facilitate data exploration and analysis. Combining these tools provides a simple, user-friendly workflow to facilitate 16S rRNA gene amplicon sequencing data analysis from raw reads to results.
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Khan, Abdur R., Wisnu A. Wicaksono, Natalia J. Ott, Amisha T. Poret-Peterson, and Greg T. Browne. "Random forest analysis reveals taxa predictive of Prunus replant disease in peach root microbiomes." PLOS ONE 17, no. 10 (October 13, 2022): e0275587. http://dx.doi.org/10.1371/journal.pone.0275587.
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Successive plantings of Prunus species produce suboptimal growth and yield in many California soils due to a poorly understood soilborne disease complex, Prunus replant disease (PRD). We explored the hypothesis that PRD is mediated by microbial taxa in roots of Nemaguard peach, a rootstock for almond and other stone fruits. In a greenhouse bioassay, portions of 10 replant soils were treated with fumigation or pasteurization or left untreated as a control before being planted with peach seedlings. Ten weeks after planting, seedlings were considered PRD-affected if their top fresh weights in the control were significantly reduced, compared to the weights in pasteurization and fumigation treatments; plants with equivalent top weights in all treatments were considered to be non-affected. The roots were washed from the soil, frozen, extracted for total DNA, and used for metabarcoding of rRNA gene amplicons from bacteria, fungi, and oomycetes. High-throughput amplicon sequencing revealed that root microbial community shifts resulted from preplant treatments, and specific taxa were associated with PRD induction among controls. Random forest (RF) analysis discriminated effectively between PRD-affected and non-affected root communities. Among the 30 RF top-ranked amplicon sequence variant (ASV) predictors, 26 were bacteria, two were oomycetes, and two were fungi. Among them, only Streptomyces scabiei, Steroidobacter denitrificans, Streptomyces bobili, and Pythium mamillatum had root abundances ≥5% that were either associated positively (former two ASVs) or negatively (latter two) with PRD. Thus, our findings were consistent with microbial mediation of PRD in roots and suggested taxa that may be involved in the mediation.
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Pepe-Ranney, C., C. Keyser, J. K. Trimble, and B. Bissinger. "Surveying the Sweetpotato Rhizosphere, Endophyte, and Surrounding Soil Microbiomes at Two North Carolina Farms Reveals Underpinnings of Sweetpotato Microbiome Community Assembly." Phytobiomes Journal 4, no. 1 (January 2020): 75–89. http://dx.doi.org/10.1094/pbiomes-07-19-0038-r.
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Farmers grow sweetpotatoes worldwide and some sub-Saharan African and Asian diets include sweetpotato as a staple, yet the sweetpotato microbiome is conspicuously less studied relative to crops such as maize, soybean, and wheat. Studying sweetpotato microbiome ecology may reveal paths to engineer the microbiome to improve sweetpotato yield, and/or combat sweetpotato pests and diseases. We sampled sweetpotatoes and surrounding soil from two North Carolina farms. We took samples from sweetpotato fields under two different land management regimes, conventional and organic, and collected two sweetpotato cultivars, ‘Beauregard’ and ‘Covington’. By comparing small subunit rRNA gene amplicon sequence profiles from sweetpotato storage root skin, rhizosphere, and surrounding soil, we found the skin microbiome possessed the least composition heterogeneity among samples, lowest alpha-diversity, and was significantly nested by the rhizosphere in amplicon sequence variant (ASV) membership. Many ASVs were specific to a single field and/or only found in either the skin, rhizosphere, or surrounding soil. Notably, sweetpotato skin enriched for Planctomycetaceae in relative abundance at both farms. This study elucidates underpinnings of sweetpotato microbiome community assembly, quantifies microbiome composition variance within a single farm, and reveals microorganisms associated with sweetpotato skin that belong to common but uncultured soil phylotypes. [Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .
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Jin, Boyang Tom, Feng Xu, Raymond T. Ng, and James C. Hogg. "Mian: interactive web-based microbiome data table visualization and machine learning platform." Bioinformatics 38, no. 4 (November 12, 2021): 1176–78. http://dx.doi.org/10.1093/bioinformatics/btab754.
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Abstract Summary Mian is a web application to interactively visualize, run statistical tools and train machine learning models on operational taxonomic unit (OTU) or amplicon sequence variant (ASV) datasets to identify key taxonomic groups, diversity trends or taxonomic composition shifts in the context of provided categorical or numerical sample metadata. Tools, including Fisher’s exact test, Boruta feature selection, alpha and beta diversity, and random forest and deep neural network classifiers, facilitate open-ended data exploration and hypothesis generation on microbial datasets. Availability Mian is freely available at: miandata.org. Mian is an open-source platform licensed under the MIT license with source code available at github.com/tbj128/mian. Supplementary information Supplementary data are available at Bioinformatics online.
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Kayikcioglu, Tunc, Jasmine Amirzadegan, Hugh Rand, Bereket Tesfaldet, Ruth E. Timme, and James B. Pettengill. "Performance of methods for SARS-CoV-2 variant detection and abundance estimation within mixed population samples." PeerJ 11 (January 26, 2023): e14596. http://dx.doi.org/10.7717/peerj.14596.
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Background The accurate identification of SARS-CoV-2 (SC2) variants and estimation of their abundance in mixed population samples (e.g., air or wastewater) is imperative for successful surveillance of community level trends. Assessing the performance of SC2 variant composition estimators (VCEs) should improve our confidence in public health decision making. Here, we introduce a linear regression based VCE and compare its performance to four other VCEs: two re-purposed DNA sequence read classifiers (Kallisto and Kraken2), a maximum-likelihood based method (Lineage deComposition for Sars-Cov-2 pooled samples (LCS)), and a regression based method (Freyja). Methods We simulated DNA sequence datasets of known variant composition from both Illumina and Oxford Nanopore Technologies (ONT) platforms and assessed the performance of each VCE. We also evaluated VCEs performance using publicly available empirical wastewater samples collected for SC2 surveillance efforts. Bioinformatic analyses were performed with a custom NextFlow workflow (C-WAP, CFSAN Wastewater Analysis Pipeline). Relative root mean squared error (RRMSE) was used as a measure of performance with respect to the known abundance and concordance correlation coefficient (CCC) was used to measure agreement between pairs of estimators. Results Based on our results from simulated data, Kallisto was the most accurate estimator as it had the lowest RRMSE, followed by Freyja. Kallisto and Freyja had the most similar predictions, reflected by the highest CCC metrics. We also found that accuracy was platform and amplicon panel dependent. For example, the accuracy of Freyja was significantly higher with Illumina data compared to ONT data; performance of Kallisto was best with ARTICv4. However, when analyzing empirical data there was poor agreement among methods and variations in the number of variants detected (e.g., Freyja ARTICv4 had a mean of 2.2 variants while Kallisto ARTICv4 had a mean of 10.1 variants). Conclusion This work provides an understanding of the differences in performance of a number of VCEs and how accurate they are in capturing the relative abundance of SC2 variants within a mixed sample (e.g., wastewater). Such information should help officials gauge the confidence they can have in such data for informing public health decisions.
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Sanchez-Cid, Concepcion, Romie Tignat-Perrier, Laure Franqueville, Laurence Delaurière, Trista Schagat, and Timothy M. Vogel. "Sequencing Depth Has a Stronger Effect than DNA Extraction on Soil Bacterial Richness Discovery." Biomolecules 12, no. 3 (February 25, 2022): 364. http://dx.doi.org/10.3390/biom12030364.
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Although Next-Generation Sequencing techniques have increased our access to the soil microbiome, each step of soil metagenomics presents inherent biases that prevent the accurate definition of the soil microbiome and its ecosystem function. In this study, we compared the effects of DNA extraction and sequencing depth on bacterial richness discovery from two soil samples. Four DNA extraction methods were used, and sequencing duplicates were generated for each DNA sample. The V3–V4 region of the 16S rRNA gene was sequenced to determine the taxonomical richness measured by each method at the amplicon sequence variant (ASV) level. Both the overall functional richness and antibiotic resistance gene (ARG) richness were evaluated by metagenomics sequencing. Despite variable DNA extraction methods, sequencing depth had a greater influence on bacterial richness discovery at both the taxonomical and functional levels. Sequencing duplicates from the same sample provided access to different portions of bacterial richness, and this was related to differences in the sequencing depth. Thus, the sequencing depth introduced biases in the comparison of DNA extraction methods. An optimisation of the soil metagenomics workflow is needed in order to sequence at a sufficient and equal depth. This would improve the accuracy of metagenomic comparisons and soil microbiome profiles.
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Aralaguppe, Shambhu G., Anoop T. Ambikan, Manickam Ashokkumar, Milner M. Kumar, Luke Elizabeth Hanna, Wondwossen Amogne, Anders Sönnerborg, and Ujjwal Neogi. "MiDRMpol: A High-Throughput Multiplexed Amplicon Sequencing Workflow to Quantify HIV-1 Drug Resistance Mutations against Protease, Reverse Transcriptase, and Integrase Inhibitors." Viruses 11, no. 9 (August 30, 2019): 806. http://dx.doi.org/10.3390/v11090806.
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The detection of drug resistance mutations (DRMs) in minor viral populations is of potential clinical importance. However, sophisticated computational infrastructure and competence for analysis of high-throughput sequencing (HTS) data lack at most diagnostic laboratories. Thus, we have proposed a new pipeline, MiDRMpol, to quantify DRM from the HIV-1 pol region. The gag-vpu region of 87 plasma samples from HIV-infected individuals from three cohorts was amplified and sequenced by Illumina HiSeq2500. The sequence reads were adapter-trimmed, followed by analysis using in-house scripts. Samples from Swedish and Ethiopian cohorts were also sequenced by Sanger sequencing. The pipeline was validated against the online tool PASeq (Polymorphism Analysis by Sequencing). Based on an error rate of <1%, a value of >1% was set as reliable to consider a minor variant. Both pipelines detected the mutations in the dominant viral populations, while discrepancies were observed in minor viral populations. In five HIV-1 subtype C samples, minor mutations were detected at the <5% level by MiDRMpol but not by PASeq. MiDRMpol is a computationally as well as labor efficient bioinformatics pipeline for the detection of DRM from HTS data. It identifies minor viral populations (<20%) of DRMs. Our method can be incorporated into large-scale surveillance of HIV-1 DRM.
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Polakova, Katerina Machova, Vojtech Kulvait, Jana Linhartova, Adela Brouckova, Simona Soverini, Monika Jaruskova, Caterina de Benedittis, et al. "Algorithms and Processing Pipeline For Error Correction and Detection Of Significant Mutations In The Kinase Domain Of BCR-ABL Analyzed By Next-Generation Sequencing: Implications For Clinical Practice Of Chronic Myeloid Leukemia." Blood 122, no. 21 (November 15, 2013): 3987. http://dx.doi.org/10.1182/blood.v122.21.3987.3987.
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Abstract High sequencing depths of NGS may cause false-positive variant calls of minor subclones (up to 10%). Errors inserted into the NGS pipeline during sample preparation and sequencing manifest by erroneous detections of mutations including point mutations. Thus, there is a need for algorithms for filtering data which occur by error from relevant biological information. We developed algorithms and a processing pipeline for error correction and detection of significant mutations after NGS of BCR-ABL kinase domain (KD). We validated our algorithms on the retrospective NGS analysis of 135 samples from 15 CML patients in chronic phase (median 8 samples per patient; range 5-19), who developed resistant mutations (confirmed by Sanger Sequencing, SS) during 2-4 lines of therapy. Amplicon libraries were prepared using reverse transcription and 2-step PCR. The second PCR was performed partly using fusion-primers designed within the IRON-II study research consortium (Roche Applied Science) which tested 4 overlapping amplicons and partly using alternative in-house set of fusion-primers that we have developed upfront and which utilized 3 overlapping amplicons covering the KD coding region. Key concept of our error control algorithm was to apply statistics used for bacterial mutation rate prediction, Lea-Coulson probability distribution (Lea and Coulson, J of Genet 1949), to distinguish sequencing pipeline errors from biologically relevant mutations. We postulated that spontaneous mutations in bacteria are similar phenomena as enzyme errors in vitro. In both processes there are new generations of bacteria or transcripts in which mutations or errors replicate exponentially. The error rate distributions based on analysis of c-ABL kinase domain of healthy donors (n=24) were fitted to Lea-Coulson distribution. From this analysis we derived, for each type of single nucleotide substitution, estimated thresholds based on which a particular mutation may be called significant by a self-developed statistical test. We cross-checked our results with results of standard Roche pipeline including GS Amplicon Variant Analyzer. Table 1 summarizes the estimated thresholds to be applied for transitions and transversions. Higher frequency of errors was found in case of using a 3-amplicon assay in comparison to a 4-amplicon assay. The PCR products in the 3-amplicon assay are 71 bp longer on average than in the 4-amplicon assay, thus the error frequency distribution may be dependent on the length of the sequence amplified. Using our algorithm we processed NGS data and reported significant mutations. Overall, no significant mutation that caused resistance during the treatment was detected at the time of diagnosis. During 1st line imatinib treatment 10 resistant mutations in 9 patients were detected as significant 2-5 months earlier than by SS. At the time of therapy switchover, in 3 patients the algorithm already detected minor populations of one of significant mutations F317L, T315I and M351T, while SS did not. These mutations manifested after the therapy switchover and caused treatment failure. After the therapy switch, baseline mutations were still significantly detectable by our algorithm in NGS data, but not by SS in 7 patients who achieved at the time of the analysis PCgR and MMR. In 5 patients, who subsequently failed therapy after switchover, resistant mutations were significantly detected by our algorithm in NGS data 2-9 months earlier than by SS. New minor mutations were revealed by NGS after the therapy switch in 8 patients.Table 1TRANSITIONS (%)TRANSVERSIONS (%)P valueA/GG/AT/CC/TA/C+C/A+T/A+A/T+T/G+G/T+C/G+G/C3-amplicon assay0.0112.24.5311.84.770.570.053.031.032.931.100.134-amplicon assay0.015.171.934.501.930.130.051.200.431.030.430.03 Since enzymes create errors during reverse transcription, 2-step PCR and sequencing process, the error correction is an essential part of the bioinformatics pipeline for relevant interpretation of BCR-ABL KD mutations detected with the highly sensitive NGS assay. Our validated algorithm and processing pipeline for significant mutation evaluations from NGS data is helpful for future clinical practice as it filters errors and allows reporting only significant mutations. This avoids false-positive results and misleading interpretations which may negatively influence treatment management of CML patients. Supported by IGANT11555 and NT13899 Disclosures: Machova Polakova: Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Soverini:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; ARIAD: Consultancy. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Martinelli:NOVARTIS, BMS(Consultancy and speaker bureau), PFIZER, ARIAD ( Consultancy): Consultancy, Speakers Bureau. Kohlmann:MLL Munich Leukemia Laboratory: Employment; Roche Diagnostics: Honoraria. Klamova:Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding.
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Gerber, Tiemo S., Arno Schad, Nils Hartmann, Erik Springer, Ulrich Zechner, and Thomas J. Musholt. "Targeted next-generation sequencing of cancer genes in poorly differentiated thyroid cancer." Endocrine Connections 7, no. 1 (January 2018): 47–55. http://dx.doi.org/10.1530/ec-17-0290.
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Poorly differentiated thyroid carcinoma (PDTC) is a rare malignancy with higher mortality than well-differentiated thyroid carcinoma. The histological diagnosis can be difficult as well as the therapy. Improved diagnosis and new targeted therapies require knowledge of DNA sequence changes in cancer-relevant genes. The TruSeq Amplicon Cancer Panel was used to screen cancer genomes from 25 PDTC patients for somatic single-nucleotide variants in 48 genes known to represent mutational hotspots. A total of 4490 variants were found in 23 tissue samples of PDTC. Ninety-eight percent (4392) of these variants did not meet the inclusion criteria, while 98 potentially pathogenic or pathogenic variants remained after filtering. These variants were distributed over 33 genes and were all present in a heterozygous state. Five tissue samples harboured not a single variant. Predominantly, variants in P53 (43% of tissue samples) were identified, while less frequently, variants in APC, ERBB4, FLT3, KIT, SMAD4 and BRAF (each in 17% of tissue samples) as well as ATM, EGFR and FBXW7 (each in 13% of tissue samples) were observed. This study identified new potential genetic targets for further research in PDTC. Of particular interest are four observed ERBB4 (alias HER4) variants, which have not been connected to this type of thyroid carcinoma so far. In addition, APC and SMAD4 mutations have not been reported in this subtype of cancer either. In contrast to other reports, we did not find CTNNB1 variants.
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Grossmann, Vera, Alexander Kohlmann, Claudia Haferlach, Hans-Ulrich Klein, Martin Dugas, Frank Dicker, Beray Kazak, et al. "Next-Generation Sequencing (NGS) in CMML, MDS and AML Detects Molecular Mutations in Oncogenes and Allows the Identification of Balanced Chromosomal Abnormalities with Extraordinary Sensitivity and Specificity." Blood 114, no. 22 (November 20, 2009): 144. http://dx.doi.org/10.1182/blood.v114.22.144.144.
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Abstract Abstract 144 PicoTiterPlate (PTP) pyrosequencing allows the detection of low-abundance oncogene aberrations in complex samples even with low tumor content. Here, we compared deep sequencing data of two Next-Generation Sequencing (NGS) assays to detect molecular mutations using a PCR-based strategy and, in addition, to uncover inversions, translocations, and insertions in a targeted sequence enrichment workflow (454 Life Sciences, Roche Diagnostics Corporation, Branford, CT). First, we studied 95 patients (CMML, n=81; AML, n=6; MDS, n=3; MPS, n=3; ET, n=2) using the amplicon approach and investigated seven candidate genes with relevance in oncogenesis of myeloid malignancies: TET2, RUNX1, JAK2, MPL, KRAS, NRAS, and CBL. 43 primer pairs were designed to cover the complete coding regions of TET2, RUNX1 (beta isoform), and hotspot regions of the latter genes. In total, 4128 individual PCR reactions were performed with DNA isolated from bone marrow mononuclear cells, followed by product purification, fluorometric quantitation, and equimolar pooling of the corresponding 43 amplicon products to generate one single sequence library per patient. For sequencing, a 454 8-lane PTP was used applying standard FLX chemistry and representing one patient per lane. The median number of base pairs sequenced per patient was 9.23 Mb. For each amplicon a median of 840 reads was generated (coverage range: 485–1929 reads). As initial proof-of-concept analysis 27 of the 95 patients with known mutations (n=32) as detected by conventional sequencing or melting curve analyses were investigated (range of cells carrying the respective mutation: 1.1% for JAK2 V617F to 98.14% for TET2 C1464X). In all cases, 454 NGS confirmed results from routine diagnostic methods (GS Amplicon Variant Analyzer software version 2.0.01). We then investigated the remaining 69 CMML patients: In median, 2 variances (range 1–8 variances), i.e. differences in comparison to the reference sequence, per patient were detected. These variances included both point mutations in all candidate genes and large deletions (12-19 bp) in CBL, RUNX1, and TET2. Only 20/81 patients of the CMML-cohort (24.69%) were without any detectable mutation. Secondly, in a cohort of six AML bone marrow specimens a custom NimbleGen array (385K format; Madison, WI) was used to perform a targeted DNA sequence enrichment procedure. In total, capture probes spanning 1.91 Mb were designed to represent all coding regions of 92 target genes (1559 exons) with relevance in hematological malignancies (e.g. KIT, NF1, TP53, BCR, ABL1, NPM1, or FLT3). In addition, the complete genomic regions were targeted for RUNX1, CBFB, and MLL. For sequencing, 454 Titanium chemistry was applied, loading three patients per lane on a 2-lane PTP including three molecular identifiers (MIDs) each. Data analysis was performed using the GS Reference Mapper software version 2.0.01. For the enrichment assay, the median enrichment of the targeted genomic loci was 207-fold, as assessed by ligation-mediated LM-PCR. Overall, 1,098,132 reads were generated in the two lanes, yielding a total sequence length of 386,097,740 bases. In median, 96.52% of the sequenced bases mapped against the human genome, and 66.0% were derived from the customized NimbleGen array capture probes, resulting in a median coverage of 18.7-fold . With this method it was possible to detect and confirm point mutations (KIT, FLT3-TKD, and KRAS) and insertions (FLT3-ITD). Moreover, by capturing chimeric DNA fragments and generating reads mapping to both fusion partners this approach detected balanced aberrations, i.e. inv(16)(p13q22) and the translocations t(8;21)(q22;q22) or t(9;11)(p22;q23). In conclusion, both assays to specifically sequence targeted regions with oncogenic relevance on a NGS platform demonstrated promising results and are feasible. The amplicon approach is more suitable for detection of mutations in a routine setting and is ideally suited for large genes such as TET2, ATM, and NF1, which are labor-intensive to sequence conventionally. The array-based capturing assay is characterized by a complex and time-consuming workflow with low-throughput. However, the ability to detect balanced genomic aberrations which are detectable thus far only by cytogenetics and FISH has the potential to become an important diagnostic assay, especially in tumors in which cytogenetics can not be applied successfully. Disclosures: Grossmann: MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Dicker:MLL Munich Leukemia Laboratory: Employment. Kazak:MLL Munich Leukemia Laboratory: Employment. Schindela:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.
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Conley, Andrew, Huazhang Li, and Alex V. Kotlar. "Abstract 5066: Accurate detection of tumor sequence at low tumor content for MRD." Cancer Research 82, no. 12_Supplement (June 15, 2022): 5066. http://dx.doi.org/10.1158/1538-7445.am2022-5066.
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Abstract Background Accurate detection of tumor sequence when the overall tumor content (TC) of the sample is low (<0.01%) is critical for tumor-informed minimal residual disease (MRD) monitoring. Current methodology generally relies on detection of multiple low-VAF (variant allele fraction) co-mutations. At low TC and low DNA input (<30ng, 9,000 genome equivalents), many variant sites are not expected to have any tumor-derived fragments or will be only marginally above background noise. A method that does not rely on the positive identification of mutations would be useful for detecting early disease recurrence. Methods We developed a maximum likelihood (ML) method for inferring the TC of MRD samples, rather than VAF at individual sites. This method considers the frequency of non-reference reads at each variant site along with estimated error rates. To evaluate our method at low TC, we carried out spike-in simulations of MRD samples across a range of sample TC from 0 to 0.1%. For each simulation, we randomly selected a single normal from a pool of sequenced normal samples to serve as background. Using this background, we randomly selected sites and mutations to represent somatic variants, sampled the existing noise at each site, and the number of ctDNA fragments and reads given the tumor content. Intrinsic error rates were modeled as either the mean error of the sample, the per-reference base error, or the mutation error. An additional randomly selected normal sample was used for extrinsic, per-site error estimates. We created contrived tumor-informed MRD samples by serial two-fold dilution of tumor DNA in a normal background (0.2%-0.0125% sample TC), followed by amplicon sequencing and characterization by our ML method. Results In our spike-in simulations, using 32 variants sites, we achieved 99% sensitivity in detecting tumor sequence at a TC of 0.01% (average ~1 tumor fragment per site), 73% sensitivity at 0.005% (average ~0.5 tumor fragments per site), and 99.9% specificity when using extrinsic error from a normal sample. At 16 sites we achieved 95% and 68% sensitivity respectively, and 99.6% specificity. Without the extrinsic error, specificity was greatly reduced to 71% (16 sites) and 77% (32 sites) at .005% TC. This highlights the advantage of extrinsic error in estimating low TC. In our contrived samples, we found that TC estimates from our ML approach correlated well with the expected values (r = 0.99). Conclusions With our approach, we were able to accurately quantify TC in both simulated and contrived samples. In simulated samples, we can detect TC well below 0.01% with high specificity and using only 16 sites. Importantly, the TC estimation does not rely on identifying individual positive sites. While modeling the intrinsic sequencing error rates in a sample does increase our ability to characterize TC, extrinsic, per-site error rates are needed to control false positives introduced by constitutively noisy sites. Citation Format: Andrew Conley, Huazhang Li, Alex V. Kotlar. Accurate detection of tumor sequence at low tumor content for MRD [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5066.
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Hollants, Silke, Egbert J. W. Redeker, and Gert Matthijs. "Microfluidic Amplification as a Tool for Massive Parallel Sequencing of the Familial Hypercholesterolemia Genes." Clinical Chemistry 58, no. 4 (April 1, 2012): 717–24. http://dx.doi.org/10.1373/clinchem.2011.173963.
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Abstract BACKGROUND Familial hypercholesterolemia (FH) is an autosomal dominant disorder that affects cholesterol metabolism and is an important risk factor for heart disease. Three different genes were causally linked to this disorder: LDLR (low density lipoprotein receptor), APOB [apolipoprotein B (including Ag(x) antigen)], and PCSK9 (proprotein convertase subtilisin/kexin type 9). We evaluated a new amplicon preparation tool for resequencing these genes on next generation sequencing (NGS) platforms. METHODS For the 3 genes, 38 primer pairs were designed and loaded on the Fluidigm Access Array, a microfluidic array in which a PCR was performed. We amplified 144 DNA samples (73 positive controls and 71 patient samples) and performed 3 sequencing runs on a GS FLX Titanium system from Roche 454, using pyrosequencing. Data were analyzed with the SeqNext module of the Sequence Pilot software. RESULT From the 38 amplicons, 37 were amplified successfully, without any further optimization. Sequencing resulted in a mean coverage of the individual amplicons of 71-fold, 74-fold, and 117-fold for the 3 runs, respectively. In the positive controls, all known mutations were identified. In 29% of the patient samples, a pathogenic point mutation or small deletion/insertion was found. Large rearrangements were not detectable with NGS, but were picked up by multiplex ligation-dependent probe amplification. CONCLUSIONS Combining a microfluidic amplification system with massive parallel sequencing is an effective method for mutation scanning in FH patients, which can be implemented in diagnostics. For data analysis, we propose a minimum variant frequency threshold of 20% and a minimum coverage of 25-fold.
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Haruna, Ishaku Lemu, Huitong Zhou, and Jon G. H. Hickford. "Variation in bovine leptin gene affects milk fatty acid composition in New Zealand Holstein Friesian × Jersey dairy cows." Archives Animal Breeding 64, no. 1 (June 7, 2021): 245–56. http://dx.doi.org/10.5194/aab-64-245-2021.
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Abstract. Leptin is a protein hormone secreted from white adipose tissue. It regulates food/feed intake, body weight, immune function and reproduction. In our investigation, the polymerase chain reaction (PCR) amplification coupled with single-strand conformational polymorphism (SSCP) analysis was used to reveal variation in bovine leptin gene (LEP) in New Zealand (NZ) Holstein Friesian × Jersey (HF × J) dairy cows. Subsequent sequence analysis of a 430 bp amplicon spanning the entirety of exon 3 and part of the intron 2 region revealed three variant sequences (A3, B3 and C3) containing a total of five nucleotide substitutions, all of which have been reported previously. Using general linear mixed-effect model analyses, the presence of variant A3 (the most common variant) was associated with a decreased level of C15:1, C18:1 trans-11, C18:1 all trans, C18:2 trans-9, cis-12, C22:0 and C24:0 levels but increased levels of C12:1 and C13:0 iso (p<0.05). Variant B3 was associated with reduced levels of C6:0, C8:0, C11:0, C13:0 and C20:0 but increased C17:0 iso and C24:0 levels (p<0.05). Variant C3 was associated with decreased C17:0 iso levels but increased C20:0 (p<0.05) levels. In a genotype model, the A3B3 genotype was associated with increased levels of C22:0 and C24:0 but decreased C8:0, C10:0, C11:0, C13:0, C15:0 and grouped medium-chain fatty acid (MCFA) levels (p<0.05). Genotype A3C3 was found to be associated with decreased levels of C10:0, C11:0, C13:0 and grouped MCFA (p<0.05). This is the first report of findings of this kind in NZ HF × J cows, and they suggest that variation in exon 3 of bovine leptin gene could be explored as a means of decreasing the concentration of saturated fatty acids in milk.
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Iacobucci, Ilaria, Anna Ferrari, Alexander Kohlmann, Cristina Papayannidis, Claudia Venturi, Margherita Perricone, Maria Chiara Fontana, et al. "Loss of Heterozygosity At the C Wild-Type Allele of rs1042522 in the TP53 Gene Frequently Occurs During Progression of Adult BCR-ABL1 Positive Acute Lymphoblastic Leukemia (ALL)." Blood 120, no. 21 (November 16, 2012): 2497. http://dx.doi.org/10.1182/blood.v120.21.2497.2497.
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Abstract Abstract 2497 Introduction: The gene TP53 encoding the tumour suppressor protein p53 is among the most commonly mutated genes in human cancer. TP53 tumour-associated alterations often cause dramatic defects in p53 function and correlate with increased malignancy, dismal survival and resistance to treatment. In contrast, only a small fraction, if any, of the >200 single nucleotide polymorphisms (SNPs) of TP53 in human populations are expected to cause measurable perturbation of p53 function. Aim: Since the pattern, frequency and significance of TP53 aberrations and SNPs in adult BCR-ABL1 positive ALL have still to be investigated, in this study we used a massively parallel pyrosequencing technique to address these issues. Patients and methods: Forty-three adults with BCR-ABL1 positive ALL were analyzed (median age 63 years, range 18–84). Twenty-four cases (56%) were analyzed only at the time of diagnosis, four cases (9%) only at the time of relapse and fifteen cases (35%) at both time points. Massively parallel pyrosequencing in picoliter-sized wells was used to allow highly-sensitive deep-sequencing detecting molecular aberrations at a low burden rate. As part of the IRON (Interlaboratory RObustness of Next generation sequencing) II study network, we applied preconfigured plates including primers for TP53 (exons 4 to 11) and allowing the simultaneous screening of 11 patients, each being recognized by a unique molecular identifier. For each plate, after generating 88 amplicons, 8 per each patient, PCR products were purified using Agencourt AMPure XP beads and Biomek 3000 Laboratory Automation Workstation (Beckam Coulter) and quantified using the Quant-iT PicoGreen kit (Invitrogen). All amplicons were pooled in an equimolar ratio to generate one single library. Subsequent emulsion PCR and amplicon sequencing was performed according to the manufacturer's recommendations on the Genome Sequencer Junior Instrument (Roche Applied Science). Data were analyzed using the GS Amplicon Variant Analyzer software version 2.7 (Roche Applied Science). For the detection of variances, filters were set to display sequence variances occurring in more than 5% of bidirectional reads per amplicon in at least one sample. Results: On average, we generated 63,068 sequencing beads (key pass wells) per plate (range, 50,798-79,486) with a median read length range between 284 and 365 base pairs. The median number of generated reads per case was 5,413 (range, 687-9,604). The median number of reads per amplicon was as follows: exon 4: 275 (range, 0–888), exon 5: 222 (range, 0–1,013); exon 6: 316 (range, 84–854); exon 7: 317 (range, 5–720); exon 8: 313 (range, 0–784); exon 9: 215 (range, 0–785); exon 10: 328 (range, 0–826), exon 11: 447 (range, 0–1,511). Forward and reverse reads were homogeneously distributed allowing a sensitive detection of variances. In total, 8 single nucleotide variations were identified. All variances, except for one nucleotide substitution occurring at position 7576743 (GRCh37/hg19), were found to represent SNPs according to the NCBI dbSNP Build 137. They included: rs1042522 C/G (41/43, 95%) and rs1800370 A/G (1/43, 2%) in exon 4, rs1800372 A/G (2/43, 5%) in exon 6, rs1625895 A/G (42/43, 98%) in intron 6–7, rs12947788 A/G (3/43, 7%) and rs12951053 A/C (4/43, 9%) in intron 7–8 and rs1800899 C/T (1/43, 2%) in intron 9–10. Interestingly, in 2 cases (12%) loss of heterozygosity occurred at the relapse at the C wild-type allele of rs1042522 in leukemia cells. The same mechanism has been identified for one case at the wild-type allele of rs1625895 with the expansion of the variant form at relapse. Both rs1042522 and rs1625895 have been described to alter p53 functionality and increase susceptibility to cancers (Whibley et al.,2009). Although the role of rs12947788 and rs12951053 has not yet deeply investigated, in our study they were found in 3 cases that all relapsed. Conclusion: Comprehensive next generation deep-sequencing of TP53 by a screening assay set up within the IRON II study has demonstrated its ability to efficiently detect TP53 variant. The inactivation of the wild-type allelic forms of rs1042522 and rs1625895, altering the p53 functionality, may serve as an important background for leukemia progression in BCR-ABL1-positive ALL. Disclosures: Kohlmann: MLL Munich Leukemia Laboratory: Employment. Luppi:CELGENE CORPORATION: Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Baccarani:ARIAD, Novartis, Bristol Myers-Squibb, and Pfizer: Consultancy, Honoraria, Speakers Bureau. Martinelli:NOVARTIS: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.
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Vandersteen, Joshua G., Pinar Bayrak-Toydemir, Robert A. Palais, and Carl T. Wittwer. "Identifying Common Genetic Variants by High-Resolution Melting." Clinical Chemistry 53, no. 7 (July 1, 2007): 1191–98. http://dx.doi.org/10.1373/clinchem.2007.085407.
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Abstract Background: Heteroduplex scanning techniques usually detect all heterozygotes, including common variants not of clinical interest. Methods: We conducted high-resolution melting analysis on the 24 exons of the ACVRL1 and ENG genes implicated in hereditary hemorrhagic telangiectasia (HHT). DNA in samples from 13 controls and 19 patients was PCR amplified in the presence of LCGreen® I, and all 768 exons melted in an HR-1® instrument. We used 10 wild-type controls to identify common variants, and the remaining samples were blinded, amplified, and analyzed by melting curve normalization and overlay. Unlabeled probes characterized the sequence of common variants. Results: Eleven common variants were associated with 8 of the 24 HHT exons, and 96% of normal samples contained at least 1 variant. As a result, the positive predictive value (PPV) of a heterozygous exon was low (31%), even in a population of predominantly HHT patients. However, all common variants produced unique amplicon melting curves that, when considered and eliminated, resulted in a PPV of 100%. In our blinded study, 3 of 19 heterozygous disease-causing variants were missed; however, 2 were clerical errors, and the remaining false negative would have been identified by difference analysis. Conclusions: High-resolution melting analysis is a highly accurate heteroduplex scanning technique. With many exons, however, use of single-sample instruments may lead to clerical errors, and routine use of difference analysis is recommended. Common variants can be identified by their melting curve profiles and genotyped with unlabeled probes, greatly reducing the false-positive results common with scanning techniques.
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Dubagari, Nkpusireufu Gambo, B. I. Mwagu, and O. A. Ojo. "SINGLE NUCLEOTIDE POLYMORPHISM DETECTION AND SEQUENCE CHARACTERIZATION OF BETA CASEIN GENE IN BUNAJI AND FRIESIAN-BUNAJI CROSSBRED COW." FUDMA JOURNAL OF SCIENCES 6, no. 2 (May 11, 2022): 156–59. http://dx.doi.org/10.33003/fjs-2022-0602-917.
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Milk is an important food constituent in diet. This study was undertaken to detect the presence of SNP in βcasein gene of Bunaji and Friesian X Bunaji crossbred cows using sequencing. 60 animal comprising of 30 Bunaji and 30 friesian X Bunaji cows were understudy. DNA was extracted from 200 μl of the blood using high salt method. The electrophoresis revealed molecular DNA with a single band. The exon 7 of βcasein gene was amplified by PCR using published primer. 20 amplicon were sent for sequencing, 10 each for Bunaji and Friesian X Bunaji crossbred. The DNA sequence of exon 7 of beta casein gene were aligned using the MAFFT online sequence alignment tools. The results revealed, 6 polymorphic sites at exon 7 beta casein gene. The SNP were at positions; 221, 350, 381, 387, 510, 387 and 516. Four of the SNPs were nonsynonymous in nature while 2 were synonymous. A nucleotide substitution from C→A was observed in Bunaji and its crossbred counterpart resulted in substitution of amino acid proline → histidine; and C→G substitution resulting in a Serine → Arginine substitution implying that Bunaji and Friesian X Bunaji had the preponderance of A2, A1 and B variant, resulting in potentials release of bioactive peptide upon digestion of A1 and B variant. SNPs discovered in this study can be used as molecular genetic markers for marker assisted selection (MAS) to increase the rate of genetic improvement of milk production traits in Bunaji and Friesian Bunaji crossbred cows
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Nascimento, Fernanda Sayuri do, Milena Oliveira Suzuki, João Victor Taba, Vitoria Carneiro de Mattos, Leonardo Zumerkorn Pipek, Eugênia Machado Carneiro D’Albuquerque, Leandro Iuamoto, et al. "Analysis of biliary MICRObiota in hepatoBILIOpancreatic diseases compared to healthy people [MICROBILIO]: Study protocol." PLOS ONE 15, no. 11 (November 19, 2020): e0242553. http://dx.doi.org/10.1371/journal.pone.0242553.
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Background The performance of the microbiota is observed in several digestive tract diseases. Therefore, reaching the biliary microbiota may suggest ways for studies of biomarkers, diagnoses, tests and therapies in hepatobiliopancreatic diseases. Methods Bile samples will be collected in endoscopic retrograde cholangiopancreatography patients (case group) and living liver transplantation donors (control group). We will characterize the microbiome based on two types of sequence data: the V3/V4 regions of the 16S ribosomal RNA (rRNA) gene and total shotgun DNA. For 16S sequencing data a standard 16S processing pipeline based on the Amplicon Sequence Variant concept and the qiime2 software package will be employed; for shotgun data, for each sample we will assemble the reads and obtain and analyze metagenome-assembled genomes. Results The primary expected results of the study is to characterize the specific composition of the biliary microbiota in situations of disease and health. In addition, it seeks to demonstrate the existence of changes in the case of illness and also possible disease biomarkers, diagnosis, interventions and therapies in hepatobiliopancreatic diseases. Trial registration NCT04391426. Registered 18 May 2020, https://clinicaltrials.gov/ct2/show/NCT04391426.
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Schagen, Micaela, Jason Bosch, Jenny Johnson, Robbert Duker, Pedro Lebre, Alastair J. Potts, and Don A. Cowan. "The soil microbiomics of intact, degraded and partially-restored semi-arid succulent thicket (Albany Subtropical Thicket)." PeerJ 9 (October 6, 2021): e12176. http://dx.doi.org/10.7717/peerj.12176.
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This study examines the soil bacterial diversity in the Portulacaria afra-dominated succulent thicket vegetation of the Albany Subtropical Thicket biome; this biome is endemic to South Africa. The aim of the study was to compare the soil microbiomes between intact and degraded zones in the succulent thicket and identify environmental factors which could explain the community compositions. Bacterial diversity, using 16S amplicon sequencing, and soil physicochemistry were compared across three zones: intact (undisturbed and vegetated), degraded (near complete removal of vegetation due to browsing) and restored (a previously degraded area which was replanted approximately 11 years before sampling). Amplicon Sequence Variant (ASV) richness was similar across the three zones, however, the bacterial community composition and soil physicochemistry differed across the intact and degraded zones. We identified, via correlation, the potential drivers of microbial community composition as soil density, pH and the ratio of Ca to Mg. The restored zone was intermediate between the intact and degraded zones. The differences in the microbial communities appeared to be driven by the presence of plants, with plant-associated taxa more common in the intact zone. The dominant taxa in the degraded zone were cosmopolitan organisms, that have been reported globally in a wide variety of habitats. This study provides baseline information on the changes of the soil bacterial community of a spatially restricted and threatened biome. It also provides a starting point for further studies on community composition and function concerning the restoration of degraded succulent thicket ecosystems.
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Dritsoulas, Alexandros, Sheng-Yen Wu, Homan Regmi, and Larry W. Duncan. "Arthropod Community Responses Reveal Potential Predators and Prey of Entomopathogenic Nematodes in a Citrus Orchard." Agronomy 12, no. 10 (October 13, 2022): 2502. http://dx.doi.org/10.3390/agronomy12102502.
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The contributions of soil arthropods to entomopathogenic nematode (EPN) food webs are mainly studied in artificial conditions. We investigated changes in arthropod communities in a citrus orchard following soil inundation with Steinernema feltiae or Heterorhabditis bacteriophora. We hypothesized that arthropod taxa, which decline or increase in response to EPN augmentation, represent potential prey or predators of EPN, respectively. Soil was sampled periodically after nematodes were applied, DNA was extracted from organisms recovered by sucrose centrifugation, libraries were prepared, and the ITS2 and CO1 genes were sequenced using Illumina protocol. Species from 107 microarthropod (mites and collembola) families and 121 insect families were identified. Amplicon sequence variant (ASV) reads for H. bacteriophora were less than 10% of those for S. feltiae three days after inundation, whereas microarthropod ASVs were double in plots with H. bacteriophora compared to those with S. feltiae. Significantly fewer microarthropod and insect reads in S. feltiae compared to untreated plots suggest the possibility that S. feltiae preyed on mites and Collembola in addition to insects. The responses over time of the individual microarthropod species (MOTU) suggest that regulation (up or down) of these EPN resulted from a cumulative response by many species, rather than by a few key species.
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Burns, Adam, Ruth Clifford, Helene Dreau, Chris S. R. Hatton, Shirley Henderson, Jenny Taylor, and Anna Schuh. "Targeted Gene Profiling Identifies Differential Genetic Make-up Depending On Chronic Lymphocytic Leukaemia Subtype." Blood 120, no. 21 (November 16, 2012): 1383. http://dx.doi.org/10.1182/blood.v120.21.1383.1383.
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Abstract Abstract 1383 Explorative genome-wide next-generation sequencing of leukaemias and lymphomas has revealed a wide spectrum of acquired mutations and considerable tumour heterogeneity that might be responsible for disease initiation, resistance to treatments and relapse. There is, therefore, a clinical need to identify these genetic abnormalities in a diagnostic setting. Here, we present the development and validation of a targeted next generation mutation analysis tool. To compare the distribution pattern of genetic abnormalities in chronic lymphocytic leukemia (CLL), we performed targeted deep sequencing on CLL samples using a TruSeq custom designed targeted amplicon assay (TSCA, Illumina). We reveal differential mutation distribution patterns depending on clinical CLL subgroups. The TSCA panel was designed to amplify 21 genes (table 1) with known or suspected links to either the development of CLL or as response predictors, including TP53, SF3B1 (Puente, Nature, 2011; Quesada et al, 2012) and NOTCH1 (Rossi, Blood, 2012). Where genes have known mutational hotspots in CLL, only these regions were included in our panel, for example exons 5–8 of TP53. For genes such as MAP2K1, where mutations are distributed throughout the coding region, every exon was targeted. In total, we were able to design an amplicon panel able to cover 99% of our desired 36,035bp target region. Table 1. List of genes included in CLL custom amplicon panel ASXL1 ATM CHD2 DDX3X FBXW7 HMCN1 IRF4 KLHL6 LRP1B MAP2K1 MAPK1 MED12 NOTCH1 PCLO POT1 SAMHD1 SF3B1 TP53 XPO1 ZFPM2 ZMYM3 In order to validate our approach, we used samples previously subjected to whole genome sequencing as controls. Of the 13 individual mutations in the control cohort, we were successfully able to detect 10 (77%) with our custom assay to an average depth of 1380x. A 19bp deletion in TP53 failed to be picked up by the variant calling software, and 2 point mutations in ATM were not detected due to the targeted nature of the assay. There was a single false positive mutation across all samples in ZFPM2, caused by a sequencing error in a homopolymer region. The sample group consisted of 45 representative CLL cases, split into two cohorts. The first cohort consisted of 11 cases that have yet to receive any treatment, whilst the second cohort comprised 34 relapsed/refractory cases. Analysis of further samples is in progress. We performed library preparation according to the manufacturers instructions. Each sample was dual indexed with two 8bp “barcodes” prior to equimolar pooling, and the final pooled library was processed on an Illumina MiSeq instrument using the TruSeq 2×150bp paired end sequencing protocol. The run produced 1.6Gb of passed filter sequence data, with 92.8% of above the quality threshold of Q30. The average depth of coverage across all samples was 849x. Primary analysis of the sequencing data was performed using the cloud based data analysis package from Illumina, which carried out the alignment and variant calling. A conservative quality score threshold of >99 was set, with all variants above this carried forward for further analysis. Our custom amplicon panel detected mutations in 35 of the samples, comprising 8 indels and 45 point mutations. Of the 54 mutations, 40 were missense, 8 were frame-shifts, 1 was a nonsense mutation and 5 are predicted to have functional effects on splicing domains. The most frequently mutated gene was TP53, followed by SF3B1, PCLO and NOTCH1 (figure 1). Fig 1 Frequency of genes with somatic mutations in our CLL cohort. Fig 1. Frequency of genes with somatic mutations in our CLL cohort. Importantly, there was good correlation between mutation allele frequencies from whole genome sequencing, targeted deep sequencing and TSCA, demonstrating that the high sensitivity of large-scale genome sequencers can be reliably applied in a diagnostic setting. We describe mutation hotspots and mutation distribution patterns and link them to clinical behaviour. For example: SF3B1 mutations occurred in 15% of patients and were linked to reduced progression free survival. In conclusion, our technique allows for rapid mutation detection of the most frequently mutated genes in CLL. Further refinements in amplicon design and variant calling will lead to added precision. TSCA design and validation for other haematological diseases is in progress. Disclosures: No relevant conflicts of interest to declare.
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Ansorge, Rebecca, Giovanni Birolo, Stephen A. James, and Andrea Telatin. "Dadaist2: A Toolkit to Automate and Simplify Statistical Analysis and Plotting of Metabarcoding Experiments." International Journal of Molecular Sciences 22, no. 10 (May 18, 2021): 5309. http://dx.doi.org/10.3390/ijms22105309.
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The taxonomic composition of microbial communities can be assessed using universal marker amplicon sequencing. The most common taxonomic markers are the 16S rDNA for bacterial communities and the internal transcribed spacer (ITS) region for fungal communities, but various other markers are used for barcoding eukaryotes. A crucial step in the bioinformatic analysis of amplicon sequences is the identification of representative sequences. This can be achieved using a clustering approach or by denoising raw sequencing reads. DADA2 is a widely adopted algorithm, released as an R library, that denoises marker-specific amplicons from next-generation sequencing and produces a set of representative sequences referred to as ‘Amplicon Sequence Variants’ (ASV). Here, we present Dadaist2, a modular pipeline, providing a complete suite for the analysis that ranges from raw sequencing reads to the statistics of numerical ecology. Dadaist2 implements a new approach that is specifically optimised for amplicons with variable lengths, such as the fungal ITS. The pipeline focuses on streamlining the data flow from the command line to R, with multiple options for statistical analysis and plotting, both interactive and automatic.
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Lodé, Laurence, Audrey Ménard, Marion Loirat, Maxime Halliez, Steven Richebourg, Pascaline Talmant, Catherine Godon, et al. "Backtracking Subclonal Mutations Of TP53 In Myelodysplasia (MDS) With Del(5q) With Next-Generation Sequencing (NGS)." Blood 122, no. 21 (November 15, 2013): 520. http://dx.doi.org/10.1182/blood.v122.21.520.520.
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Abstract Landscape analyses of mutational patterns have shown that virtually all myelodysplastic syndromes (MDS) harbor somatic mutations in >80% of cases. These molecular alterations provide useful clonality markers with a potential for early diagnosis of MDS when only cytopenia without marked dysplasia is observed. These markers have been proposed as future prognostic tools to guide therapeutic strategies (Bejar et al., 2011; Itzykson et al, 2013; Mufti et al 2013). Mutational analysis is finally a good way to track disease complexity by deciphering oligoclonality in MDS and better understand clonal evolution. Alterations in the TP53 gene are the most common cause of tumor escape from apoptosis. The aim of this study was to identify TP53 mutations in consecutive samples of lower-risk MDS (IPSS ≤1) with del(5q)obtained at follow-up or progression after sequential classical treatments. Next-generation sequencing (NGS) was used to backtrack the mutant clone(s) identified in late samples. The study was performed both by conventional Sanger sequencing and NGS on a GS Junior Instrument (Roche Applied Science, Mannheim, Germany). For each sample, eight exons (4-11) were amplified from 320 ng of DNA with preconfigured primer plates provided within the IRON II study network. PCR reactions were performed using the FastStart High Fidelity PCR System kit (Roche Applied Science). After double purification with Agencourt AMPure XP beads (Beckman Coulter, Miami, FL), exon-specific amplicon pools were generated and quantified using the Quant-iT™ Broad-Range PicoGreen DNA Assay Kit (Invitrogen, Carlsbad, CA). Emulsion PCR was performed with GS Junior emPCR Reagents (Lib-A) (Roche Applied Science) using 5 x 106 beads at a copy per bead ratio of 0.6. Finally, a fraction of 5-7% enriched beads was loaded on GS Junior Titanium sequencing PicoTiterPlate kit (Roche Applied Science). Data were analyzed for sequence alignment and variant detection using the GS Junior Sequencer and GS Amplicon Variant Analyzer softwares, versions 2.7 and 2.9 (Roche Applied Science). The results were further processed using the Sequence Pilot software version 4.0.1 (JSI Medical Systems, Kippenheim, Germany). The sensitivity of variant detection was set to a lower limit of >1% for bidirectional reads. This threshold was chosen according to a recent study investigating the assay's lower limit of detection (Grossmann et al., 2013), thus underlining the strength of NGS to identify subclones at a low frequency, not detectable by conventional Sanger analysis. A total of 89 DNA samples were extracted from the cytogenetics pellets of a cohort of 40 MDS with del(5q). TP53 mutation analysis was performed on 40 initial and 49 follow-up or progression samples including serial samples for 23 subjects. The depth of coverage was at least 500X and up to 8,444X per amplicon. Of those samples obtained and analysed at time of last follow-up or progression, 14 (61%) had TP53 mutations, mostly in the DNA-binding domain. Performing backtracking on previously collected serial samples, TP53 mutations were retrieved by NGS in 43% of initial samples (n=6), which is different from what was previously described by Jädersten et al (2011). A complete scenario of clonal evolution was retrieved in 11 cases, evidenced by TP53 mutations and/or cytogenetics. These were always consecutive to treatment with lenalidomide, yet 6 of the 12 cases without clonal evolution were also consecutive lenalidomide. Figure 1 provides the example of a complete follow-up including nine time points. More correlation with treatment will be provided. Although lenalidomide remains the treatment of choice for MDS with del(5q), resistant subclones may survive and culminate even following therapy initiation. This theory was recently suggested by Landau et al. in CLL (2013) and our test results support this. Early detection of emerging subclones could lead to initiation of alternative treatment, and we thus propose that a monitoring of TP53 alleles is performed annually after the onset of therapy for MDS using NGS. Figure 1. Figure 1. Disclosures: Kohlmann: MLL Munich Leukemia Laboratory: Employment. Moreau:CELGENE: Honoraria, Speakers Bureau; JANSSEN: Honoraria, Speakers Bureau.
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Walter, Christiane, Zhenyu Xu, Martin Zimmermann, Dirk Reinhardt, and Nils Von Neuhoff. "Amplicon Based Panel Targeted Resequencing Identified ZRSR2 As a Potential New Favorable Marker in Pediatric AML." Blood 128, no. 22 (December 2, 2016): 2905. http://dx.doi.org/10.1182/blood.v128.22.2905.2905.
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Abstract Introduction: Acute myeloid leukemia (AML) is one of the most threatening malignancies in children and adolescents. The accumulation of mutations in leukemia stem cells (LSC) is believed to lead to the development of leukemia. Cyto- and molecular genetics already identified several aberrations which are relevant in leukemogenesis, prognosis and therapy. Nevertheless, the molecular landscape and clonal evolution of AML and its clinical relevance, especially for pediatric patients, is not yet well described. Next Generation Sequencing (NGS) as an emerging sequencing technology provides the possibility to generate sequence data of high quality and detect genetic aberrations in a minimum of time. The aim of this study was to apply amplicon based panel targeted resequencing by using the TruSight Myeloid Panel (Illumina) to identify variants in 54 genes. Methods Patients: In total 150 samples derived from pediatric patients diagnosed with AML at the time of initial diagnosis or relapse were analysed regarding their mutational profile. All patients treated according to the AML-BFM therapy protocols (n=103) were chosen to determine the potential impact in prognosis. Sequencing and analysis of variants Sequencing with the TruSight Myeloid panel (Illumina) was performed on a MiseqDX. The sequencing panel is designed to identify somatic mutations associated with myeloid malignancies in 54 genes. To define variants, we excluded intronic, synonymous and variants with an allele frequency below 5% and a read depth below 50 reads. False positive variants were excluded by including healthy donors and reference samples. Variants were detected and analysed using Variant studio (Illumina) and Sophia DDM (Sophia Genetics). Almost all variants were detectable in both software, although great insertions and deletions were detectable only by Sophia DDM. Results In the cohort of 150 patients, we detected 408 mutations in the 54 genes included in the panel (fig. 1). 26% of the patients showed more than 4, 24% 3, 24% 2, 17% 1 and 9% of the patients showed 0 mutations. Four and more mutations occurred mostly in AML FAB M1 (n=17) and patients with a complex karyotype (n=6). Treatment related AML show less mutations compared to primary AML. Within the group of patients treated according the 1st line AML-BFM protocol (n=103), CEBPA, FLT3, KIT, NRAS, KRAS, NPM1 or WT1 mutations did not have prognostic relevance. Interestingly, we were able to detect mutations in ZRSR2 in 21 patients in total (SNVs in 6, InDels in 9 and splice acceptor variants in 6 patients). 15 patients were part of the group of patients who were treated according to the 1st line AML-BFM protocol. ZRSR2 encodes an essential splicing factor and the encoded protein associates with the U2 auxiliary factor heterodimer and may play a role in network interactions during spliceosome assembly [RefSeq 2008]. The presence of a ZRSR2 mutation seems to be associated with better EFS and lower cumulative incidence of relapse, respectively (fig.2). Even if patients with favourable cytogenetics were excluded, patients with mutated ZRSR2 might have better EFS (fig.2). Conclusions: Amplicon based panel targeted resequencing with the TruSight Myeloid panel provides the possibility to detect mutations in 54 genes associated to myeloid malignancies within 3 days. This will enable a faster and possible more precise characterisation of pediatric AML, either for risk group stratification or the addition of more specific treatment options. Due to the limited number of patients, the results concerning the prognostic relevance of ZRSR2 need to be confirmed in a larger patient group. Table patient characteristics Table. patient characteristics Figure 1 Number of variants detected in 54 genes Figure 1. Number of variants detected in 54 genes Figure 2 Event-free survival (EFS) and cumulative incidence for relapse for patients showing no mutation (blue) or mutations (red) in ZRSR2. Figure 2. Event-free survival (EFS) and cumulative incidence for relapse for patients showing no mutation (blue) or mutations (red) in ZRSR2. Disclosures Reinhardt: Jazz Pharma: Other: Travel Accomodation; Celgene: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees.
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Lana, Tobia, Paola de Lorenzo, Silvia Bresolin, Ilaria Bronzini, Monique L. Den Boer, Helene Cave, Eva Fronkova, et al. "Refinement of IKZF1 Genomic Status in Pediatric Philadelphia Positive Acute Lymphoblastic Leukemia." Blood 124, no. 21 (December 6, 2014): 3785. http://dx.doi.org/10.1182/blood.v124.21.3785.3785.
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Abstract Background: IKZF1 deletions are the most frequent secondary genetic alterations with prognostic impact in pediatric Philadelphia-positive B-cell precursor acute lymphoblastic leukemia (Ph+ BCP-ALL). Studies of IKZF1 status have mainly focused on deletions of the gene whereas very little is known about other genetic alterations in the IKZF1 locus. Aim: Using an amplicon deep sequencing approach to investigate IKZF1 mutations occurrence in pediatric Ph+ BCP-ALL reported as IKZF1wildtype (WT) for deletions by MLPA analysis. Methods: Six European centres participated in this study with a total of 98 patients.The 454 GS Junior system (Roche) was used to sequence the coding regions of IKZF1, Exon1 - Exon7 (Mullighan C. et al, 2009). An average depth of 250 reads (minimum of 102 reads, maximum of 853 reads) per amplicon was achieved. Quality control analysis excluded variants coverage of <50 reads in both forward and reverse directions. Variant analyses were performed using Amplicon Variant Analyzer (AVA) software (Roche). Variants at intronic regions or in homopolymeric stretches were excluded from further analysis, and a cut-off ≥10% mutated reads was applied to define mutations. Finally, variants described as SNPs or silent mutations were excluded. Results: In total, 882 amplicons were screened obtaining 337 variants. Of these, 14 variants corresponding to mutations with predicted deleterious impact on the function of IKZF1 passed the filters of our analysis pipeline. Twelve out of 98 (12%) IKZF1 non-deleted patients were mutated and 2 of them carried double mutations. Both cases showed different mutant allele frequencies of the 2 mutations, suggesting the presence of leukemic sub-populations with different IKZF1 status. Mutations can be subdivided in 2 categories: 5 miss-sense point mutations localized at the DNA binding domain with a dominant negative effect and 7 frameshift mutations leading to haploinsufficiency and impairing also the dimerization activity of IKZF1. The 5 point mutations were located in exon4 and 4 out of 5 were localized in the coding region of zinc finger 2 (ZF2), essential for DNA-protein interaction. Three patients carried the same c.698A>G substitution, that caused the change of Asparagine 159 into Serine. The 7 frameshift mutations (2 deletions, 4 insertions and 1 InDel) were identified all along the IKZF1locus. These nucleotide variations caused a shift in the reading frame and a consequent formation of premature stop codons depleting the C-terminal dimerization domain. Six out of 12 patients carrying IKZF1 mutations experienced an event (either relapse or death in CCR): 6 patients were treated in the pre-TKI (tyrosine kinase inhibitor) era and had 5 events while the remaining 6 patients received imatinib and showed a more favourable outcome with only one patient who died in CCR. This trend is in line with the findings recently reported by Van der Veer et al. (2014). In this study on IKZF1 deletions were identified in 126 of 191 patients, while 65 were defined IKZF1 WT on the basis of deletions analysis alone. Our study revealed that 6 of these 65 IKZF1 WT patients carried IKZF1 mutations (3 in the pre-TKI and 3 in the EsPhALL cohort). The outcome of mutated patients and deleted patients taken together (IKZF1 aberrant) is consistent with results reported in the Van der Veer study, both for the pre-TKI cohort [(4-year disease free survival (DFS) was 28.1% [SE 6.4] for IKZF1 aberrant vs 64.3% [SE 9.7] for IKZF1 WT, P=0.0036); Van der Veer study (30.0% [SE 6.8] for IKZF1 deleted vs 57.5% [SE 9.4] for IKZF1 WT, P=0.013)] and for the EsPhALL cohort [(4-year DSF was 55.7% [SE 6.8] for IKZF1 aberrant vs 59.8% [SE 11.6] for IKZF1 WT (P=0.348); Van der Veer study (4-year DFS was 53.6 [SE 7.0] for IKZF1 deleted vs 63.1 [SE 11.0] for IKZF1WT, P=0.1676]. Conclusion: This work revealed IKZF1 mutations in >10% of Ph+ BCP-ALL pediatric patients previously classified as IKZF1 WT on the basis of deletions analysis. Our results, together with data of IKZF1 deletions, highlight a 70-75% incidence of IKZF1 aberrations in pediatric Ph+ patients. Exon4 carried 9 of 14 aberrations: 7 point mutations and 2 deletions, suggesting a hotspot region for somatic mutations acquisition. The outcome of mutated patients is in line with the data previously published on the impact of IKZF1 deletions in pre and post TKI cohorts, suggesting a similar impact of mutations and deletions on clinical outcome of pediatric Ph+ patients. Disclosures No relevant conflicts of interest to declare.