Academic literature on the topic 'Amplicon sequence variant'

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.

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.

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.

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.

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.

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.

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.

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.

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.

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&gt;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&gt;G. The homozygotes p.G542X, c.2789 + 5G&gt;A, and c.3849 + 10kbC&gt;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.

ABSTRACT BACKGROUND: Particulate matter (PM) exposure has been linked to the exacerbation of respiratory and cardiovascular conditions as well as to adverse effects on fetal growth. To link the cross-talk that might occur between respiratory system and placenta after PM exposure, it has been proposed a novel mechanism of cell to cell communication mediated by extracellular vesicles (EVs). EVs are involved in both biological and pathological processes including pregnancy state. As PM interacts firstly with the nares, bacterial nasal microbiota (bNM) is one of the first compartments hit by PM exposure. This interaction might lead to structural and functional modifications within the bNM, which could cause variations within the EVs signaling network, which might lead to an improper immune response to PM stimuli. AIM: The main aim of the project was to identify how PM exposure might modify the homeostasis and composition of whole EV signaling network and bNM and the leading to a possible impact on newborn development. Subject recruitment: 518 volunteer pregnant women were enrolled during the 11th week of pregnancy at the ‘Clinica Mangiagalli’-Fondazione IRCCS Ca’ Granda – Ospedale Maggiore Policlinico, Milan, Italy. Among them, a group of subjects composed by 65 pregnant women, who agreed to participate to a more complex study protocol, was also identified. Exposure assessment, EV and Microbiota measurement: Exposure to PM concentrations was assessed using data obtained from FARM models for the whole population. In addition, individual exposure to short-term PM levels was retrieved through a personal sampler worn by the subgroup of 65 pregnant women. Plasmatic concentration and cellular origin of EVs were characterized by Nanoparticle tracking analysis (NTA) and flow cytometry, respectively. We investigated the bNM structure and characteristics of 65 pregnant women both at the enrolment (T0) and the following Monday during the cardiovascular screening (T1) through metabarcoding analysis of the V3–V4 regions of the 16s rRNA gene. Statistical analysis: Multivariable linear regression models were applied to test the associations between PM exposure (retrieved from both FARM models and personal sampler) and the majority of the collected outcomes such as maternal, foetal/newborns, and cardiovascular parameters as well as for bNM data. On the other hand, to evaluate possible associations between PM concentrations and EV characteristics negative binomial regression models for count data with over-dispersion were performed. In addition, multiple comparison method based on Benjamini-Hochberg False Discovery Rate (FDR) were applied for high number of comparisons. RESULTS In the whole population, PM10 exposure (measured at different time windows) resulted in decreased release of total amount of EVs, with the strongest effect related to concentration measured 13 weeks before the enrolment (13wks), whereas an inverse tendency was observed for exposure to PM2.5, although these associations were not significant. More reliable data on the finest fractions (PM1, PM2.5 and PM4) are given by personal sampler worn by a subgroup of women for a very short time period preceding the blood drawing (1.5 hours). As we considered this extremely acute effect, we observed a generalized increment in the EV count. Noteworthy, among the different analyzed EV subtypes, the levels of HERV-w+ EVs were the only to be increased by each tested PM10 time-lag. The same models applied on bNM data showed a reduction in terms of diversity (Shannon/Faith_pd ratio) and relative abundance of the genera Corynebacterium spp. and Staphylococcus spp. In addition, when the possible role of bNM as effect modifier between PM exposure and EVs release was investigated, we observed for the pregnant women with a balanced bNM an increment in terms of circulating EVs after daily PM stimuli. Moreover, increments of the heart rate values were observed after exposure to both PM10 and PM2.5 levels measured the day before the cardiovascular screening (Day -1). Focusing on newborn’s outcomes, decrements of the gestational age at birth were associated to PM concentrations measured throughout the gestation or during the 2nd trimester. CONCLUSIONS: To our knowledge, this is the first exploring the role of bNM and the EV cross-talk in determining the effects of PM exposure levels on healthy pregnancies as well as on newborn outcomes. The results obtained so far might suggest a possible role exerted by both EV concentration and the bNM in pregnant women in mediating the effects of PM exposure.

Tetragonula carbonaria (Smith 1854) is a native Australian stingless bee, hosting a diverse range of bacterial symbionts. T. carbonaria is used as a model to explore how relationships between host insects and the microbiome occur and can be detected within a single species, shedding light on how host-microbiome associations arise and are maintained across the corbiculate bees. Host-microbiome relationships are considered through the lens of phylosymbiosis. Methods for detecting phylosymbiosis are explored; different bioinformatics and statistical techniques are compared, with implications for future studies. Bayesian modelling is used to predict possible routes of acquisition of bee symbionts.