Zeitschriftenartikel: „Massively Parallel Reporter Assay“

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Inoue, Fumitaka, und Nadav Ahituv. „Decoding enhancers using massively parallel reporter assays“. Genomics 106, Nr. 3 (September 2015): 159–64. http://dx.doi.org/10.1016/j.ygeno.2015.06.005.

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Trauernicht, Max, Miguel Martinez-Ara und Bas van Steensel. „Deciphering Gene Regulation Using Massively Parallel Reporter Assays“. Trends in Biochemical Sciences 45, Nr. 1 (Januar 2020): 90–91. http://dx.doi.org/10.1016/j.tibs.2019.10.006.

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Avramopoulos, Dimitrios, Leslie Myint, Kasper Hansen, Ruihua Wang, Leandros Boukas und Loyal Goff. „SA131A MASSIVELY PARALLEL REPORTER ASSAY FOR VARIANTS ASSOCIATED WITH SCHIZOPHRENIA AND ALZHEIMER'S DISEASE“. European Neuropsychopharmacology 29 (2019): S1260—S1261. http://dx.doi.org/10.1016/j.euroneuro.2018.08.353.

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Georgakopoulos-Soares, Ilias, Naman Jain, Jesse M. Gray und Martin Hemberg. „MPRAnator: a web-based tool for the design of massively parallel reporter assay experiments“. Bioinformatics 33, Nr. 1 (06.09.2016): 137–38. http://dx.doi.org/10.1093/bioinformatics/btw584.

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Lee, Dongwon, Ashish Kapoor, Changhee Lee, Michael Mudgett, Michael A. Beer und Aravinda Chakravarti. „Sequence-based correction of barcode bias in massively parallel reporter assays“. Genome Research 31, Nr. 9 (20.07.2021): 1638–45. http://dx.doi.org/10.1101/gr.268599.120.

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Massively parallel reporter assays (MPRAs) are a high-throughput method for evaluating in vitro activities of thousands of candidate cis-regulatory elements (CREs). In these assays, candidate sequences are cloned upstream or downstream from a reporter gene tagged by unique DNA sequences. However, tag sequences may themselves affect reporter gene expression and lead to major potential biases in the measured cis-regulatory activity. Here, we present a sequence-based method for correcting tag-sequence-specific effects and show that our method can significantly reduce this source of variation and improve the identification of functional regulatory variants by MPRAs. We also show that our model captures sequence features associated with post-transcriptional regulation of mRNA. Thus, this new method helps not only to improve detection of regulatory signals in MPRA experiments but also to design better MPRA protocols.

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Hughes, Andrew E. O., Connie A. Myers und Joseph C. Corbo. „A massively parallel reporter assay reveals context-dependent activity of homeodomain binding sites in vivo“. Genome Research 28, Nr. 10 (29.08.2018): 1520–31. http://dx.doi.org/10.1101/gr.231886.117.

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Hammelman, Jennifer, Konstantin Krismer, Budhaditya Banerjee, David K. Gifford und Richard I. Sherwood. „Identification of determinants of differential chromatin accessibility through a massively parallel genome-integrated reporter assay“. Genome Research 30, Nr. 10 (24.09.2020): 1468–80. http://dx.doi.org/10.1101/gr.263228.120.

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Maricque, Brett B., Hemangi G. Chaudhari und Barak A. Cohen. „A massively parallel reporter assay dissects the influence of chromatin structure on cis-regulatory activity“. Nature Biotechnology 37, Nr. 1 (19.11.2018): 90–95. http://dx.doi.org/10.1038/nbt.4285.

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Kalita, Cynthia A., Gregory A. Moyerbrailean, Christopher Brown, Xiaoquan Wen, Francesca Luca und Roger Pique-Regi. „QuASAR-MPRA: accurate allele-specific analysis for massively parallel reporter assays“. Bioinformatics 34, Nr. 5 (22.09.2017): 787–94. http://dx.doi.org/10.1093/bioinformatics/btx598.

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Niroula, Abhishek, Ram Ajore und Björn Nilsson. „MPRAscore: robust and non-parametric analysis of massively parallel reporter assays“. Bioinformatics 35, Nr. 24 (29.07.2019): 5351–53. http://dx.doi.org/10.1093/bioinformatics/btz591.

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Abstract Motivation Massively parallel reporter assays (MPRA) enable systematic screening of DNA sequence variants for effects on transcriptional activity. However, convenient analysis tools are still needed. Results We introduce MPRAscore, a novel tool to infer allele-specific effects on transcription from MPRA data. MPRAscore uses a weighted, variance-regularized method to calculate variant effect sizes robustly, and a permutation approach to test for significance without assuming normality or independence. Availability and implementation Source code (C++), precompiled binaries and data used in the paper at https://github.com/abhisheknrl/MPRAscore and https://www.ncbi.nlm.nih.gov/bioproject/PRJNA554195. Supplementary information Supplementary data are available at Bioinformatics online.

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Qiao, Dandi, Corwin M. Zigler, Michael H. Cho, Edwin K. Silverman, Xiaobo Zhou, Peter J. Castaldi und Nan H. Laird. „Statistical considerations for the analysis of massively parallel reporter assays data“. Genetic Epidemiology 44, Nr. 7 (18.07.2020): 785–94. http://dx.doi.org/10.1002/gepi.22337.

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Kreimer, Anat, Haoyang Zeng, Matthew D. Edwards, Yuchun Guo, Kevin Tian, Sunyoung Shin, Rene Welch et al. „Predicting gene expression in massively parallel reporter assays: A comparative study“. Human Mutation 38, Nr. 9 (09.03.2017): 1240–50. http://dx.doi.org/10.1002/humu.23197.

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Rabani, Michal, Lindsey Pieper, Guo-Liang Chew und Alexander F. Schier. „A Massively Parallel Reporter Assay of 3′ UTR Sequences Identifies In Vivo Rules for mRNA Degradation“. Molecular Cell 68, Nr. 6 (Dezember 2017): 1083–94. http://dx.doi.org/10.1016/j.molcel.2017.11.014.

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Rabani, Michal, Lindsey Pieper, Guo-Liang Chew und Alexander F. Schier. „A Massively Parallel Reporter Assay of 3′ UTR Sequences Identifies In Vivo Rules for mRNA Degradation“. Molecular Cell 70, Nr. 3 (Mai 2018): 565. http://dx.doi.org/10.1016/j.molcel.2018.04.013.

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Madan, Namrata, Andrew Ghazi, Xianguo Kong, Edward Chen, Chad Shaw und Leonard C. Edelstein. „Identification of the Genetic Variant Responsible for Variable Platelet CD36 Expression By Massively Parallel Reporter Assay“. Blood 132, Supplement 1 (29.11.2018): 520. http://dx.doi.org/10.1182/blood-2018-99-118607.

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Abstract CD36 is a platelet membrane glycoprotein whose engagement with oxidized low density lipoprotein (oxLDL) results in platelet activation. Deletion of CD36 in mice fed a high fat diet results in attenuation of the pro-thrombotic state and platelet hyper-activity. The CD36 gene has been associated with platelet count, platelet volume, as well as lipid levels and CVD risk. Platelet CD36 expression is highly variable with 3-8% of non-white populations displaying a complete lack of expression. Platelet CD36 expression levels have been shown to be associated with both the platelet oxLDL response and an elevated risk of thrombo-embolism. Several genomic variants have been identified as associated with platelet CD36 levels, however none have been conclusively demonstrated to be causative. We have previously reported platelet expression Quantitative Trait Loci (eQTLs) that associate single nucleotide polymorphisms (SNPs) with platelet RNA levels, indicating genetic influence on gene expression. CD36 is one of the 612 platelet-expressed RNAs whose abundance has significant genotypic associations, suggesting a mechanism by which CD36 level is regulated. 86 eQTL SNPs located within a +/- 100kb window of the CD36 gene are associated with platelet CD36 mRNA levels at a significance of P<1x10-6, spanning a range of 125kb. Rather than prioritizing these 86 variants for functional testing based on genomic annotation, we opted to use an approach termed a Massively Parallel Reporter Array (MPRA), in which all 86 SNPs were tested simultaneously. A library of plasmids was generated in which both alleles of the variants including 150bp of surrounding genomic context were cloned 5' to a minimal promoter and unique barcode. For statistical robustness, 40 barcodes were constructed per allele, resulting in a library containing a total of ~5000 plasmids that were generated and transfected into K562 erythroleukemia cells. RNA-Seq was then conducted on the transfected cells and the abundance of individual barcodes was quantified, normalized to the DNA plasmid library, and then compared across variant alleles. The MPRA analysis led to identification of 5 SNPs located within a 35kb region 5' to the CD36 transcription start site that had significantly different barcode production between alleles. These 5 SNPs are in linkage disequilibrium with one another, with variants reported to be associated with CD36 level, and with variants reported to be associated with platelet count and volume by GWAS. To verify the functionality of these variants, we conducted luciferase assays in which the genomic sequence surrounding the variants were inserted 5' to the reporter gene. The genomic fragment containing the alternate alleles of two of these SNPs, rs2366739 and rs1194196, located within 29bp of each other, demonstrated 25-40% lower transcriptional activity than the reference alleles. Next, to determine if these two variants altered the interaction of DNA-binding proteins with the locus, we conducted electromobility-shift assays (EMSA) using probes derived from both alleles of the genomic sequence. As with the MPRA and luciferase assays, the alternative alleles of rs2366739 and rs1194196 resulted in lower levels of DNA:protein complexes, indicating an altered affinity for regulatory complexes. Finally, to further confirm the role of this genomic locus in regulating CD36 expression levels, we deleted a 596bp region containing these two SNPs from the K562 erythroleukemia cell genome using CRISPR/Cas. Deletion of this region resulted in an approximately 10-fold increase in CD36 RNA in these cells, confirming a role for this loci in the regulation of CD36 expression. These data indicate that we have identified a regulatory locus for CD36 expression, which has important implications for platelet function in cardiovascular disease. These variants are located within a retrotransposon long-terminal repeat and alter a putative GATA1 binding site, suggesting that these variants may alter binding of that transcription factor to the locus. Platelet CD36 expression level has been linked to the platelet response to oxLDL and may modulate the thrombotic response in hyperlipidemic conditions. Therefore, the mechanism described here, in which a genetic variant alters platelet CD36 levels, and thus the platelet response to hyperlipidemic conditions, may serve as an important biomarker for increased thrombotic potential. Disclosures No relevant conflicts of interest to declare.

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Kheradpour, P., J. Ernst, A. Melnikov, P. Rogov, L. Wang, X. Zhang, J. Alston, T. S. Mikkelsen und M. Kellis. „Systematic dissection of regulatory motifs in 2000 predicted human enhancers using a massively parallel reporter assay“. Genome Research 23, Nr. 5 (19.03.2013): 800–811. http://dx.doi.org/10.1101/gr.144899.112.

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Melnikov, Alexandre, Anand Murugan, Xiaolan Zhang, Tiberiu Tesileanu, Li Wang, Peter Rogov, Soheil Feizi et al. „Systematic dissection and optimization of inducible enhancers in human cells using a massively parallel reporter assay“. Nature Biotechnology 30, Nr. 3 (26.02.2012): 271–77. http://dx.doi.org/10.1038/nbt.2137.

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Mulvey, Bernard, Tomás Lagunas und Joseph D. Dougherty. „Massively Parallel Reporter Assays: Defining Functional Psychiatric Genetic Variants Across Biological Contexts“. Biological Psychiatry 89, Nr. 1 (Januar 2021): 76–89. http://dx.doi.org/10.1016/j.biopsych.2020.06.011.

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Eapen, Amy, Xiaoming Lu, Carmy Forney, Sreeja Parameswaran, John Ray, Matthew Weirauch und Leah Kottyan. „Massively Parallel Reporter Assays (MPRAs) Identify Allelic Transcriptional Dysregulation in Atopic Dermatitis“. Journal of Allergy and Clinical Immunology 145, Nr. 2 (Februar 2020): AB197. http://dx.doi.org/10.1016/j.jaci.2019.12.289.

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Kinney, Justin B., und David M. McCandlish. „Massively Parallel Assays and Quantitative Sequence–Function Relationships“. Annual Review of Genomics and Human Genetics 20, Nr. 1 (31.08.2019): 99–127. http://dx.doi.org/10.1146/annurev-genom-083118-014845.

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Over the last decade, a rich variety of massively parallel assays have revolutionized our understanding of how biological sequences encode quantitative molecular phenotypes. These assays include deep mutational scanning, high-throughput SELEX, and massively parallel reporter assays. Here, we review these experimental methods and how the data they produce can be used to quantitatively model sequence–function relationships. In doing so, we touch on a diverse range of topics, including the identification of clinically relevant genomic variants, the modeling of transcription factor binding to DNA, the functional and evolutionary landscapes of proteins, and cis-regulatory mechanisms in both transcription and mRNA splicing. We further describe a unified conceptual framework and a core set of mathematical modeling strategies that studies in these diverse areas can make use of. Finally, we highlight key aspects of experimental design and mathematical modeling that are important for the results of such studies to be interpretable and reproducible.

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Litterman, Adam J., Robin Kageyama, Olivier Le Tonqueze, Wenxue Zhao, John D. Gagnon, Hani Goodarzi, David J. Erle und K. Mark Ansel. „A massively parallel 3′ UTR reporter assay reveals relationships between nucleotide content, sequence conservation, and mRNA destabilization“. Genome Research 29, Nr. 6 (31.05.2019): 896–906. http://dx.doi.org/10.1101/gr.242552.118.

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Hudgins, Adam D., und Yousin Suh. „O4-01-03: FUNCTIONAL NON-CODING VARIANTS AFFECTING ALZHEIMER'S DISEASE RISK IDENTIFIED BY MASSIVELY PARALLEL REPORTER ASSAY“. Alzheimer's & Dementia 14, Nr. 7S_Part_26 (01.07.2006): P1400—P1401. http://dx.doi.org/10.1016/j.jalz.2018.06.2911.

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Klein, Jason C., Vikram Agarwal, Fumitaka Inoue, Aidan Keith, Beth Martin, Martin Kircher, Nadav Ahituv und Jay Shendure. „A systematic evaluation of the design and context dependencies of massively parallel reporter assays“. Nature Methods 17, Nr. 11 (12.10.2020): 1083–91. http://dx.doi.org/10.1038/s41592-020-0965-y.

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Chen, Andy B., Kriti Thapa, Hongyu Gao, Jill L. Reiter, Junjie Zhang, Xiaoling Xuei, Hongmei Gu, Yue Wang, Howard J. Edenberg und Yunlong Liu. „38766 Massively Parallel Reporter Assay Reveals Functional Impact of 3™-UTR SNPs Associated with Neurological and Psychiatric Disorders“. Journal of Clinical and Translational Science 5, s1 (März 2021): 95. http://dx.doi.org/10.1017/cts.2021.645.

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ABSTRACT IMPACT: Screening the effect of thousands of non-coding genetic variants will help identify variants important in the etiology of diseases OBJECTIVES/GOALS: Massively parallel reporter assays (MPRAs) can experimentally evaluate the impact of genetic variants on gene expression. In this study, our objective was to systematically evaluate the functional activity of 3’-UTR SNPs associated with neurological disorders and use those results to help understand their contributions to disease etiology. METHODS/STUDY POPULATION: To choose variants to evaluate with the MPRA, we first gathered SNPs from the GWAS Catalog that were associated with any neurological disorder trait with p-value < 10-5. For each SNP, we identified the region that was in linkage disequilibrium (r2 > 0.8) and retrieved all the common 3’-UTR SNPs (allele-frequency > 0.05) within that region. We used an MPRA to measure the impact of these 3’-UTR variants in SH-SY5Y neuroblastoma cells and a microglial cell line. These results were then used to train a deep-learning model to predict the impact of variants and identify features that contribute to the predictions. RESULTS/ANTICIPATED RESULTS: Of the 13,515 3’-UTR SNPs tested, 400 and 657 significantly impacted gene expression in SH-SY5Y and microglia, respectively. Of the 84 SNPs significantly impacted in both cells, the direction of impact was the same in 81. The direction of eQTL in GTEx tissues agreed with the assay SNP effect in SH-SY5Y cells but not microglial cells. The deep-learning model predicted sequence activity level correlated with the experimental activity level (Spearman’s corr = 0.45). The deep-learning model identified several predictive motifs similar to motifs of RNA-binding proteins. DISCUSSION/SIGNIFICANCE OF FINDINGS: This study demonstrates that MPRAs can be used to evaluate the effect of non-coding variants, and the results can be used to train a machine learning model and interpret its predictions. Together, these can help identify causal variants and further understand the etiology of diseases.

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Kreimer, Anat, Zhongxia Yan, Nadav Ahituv und Nir Yosef. „Meta‐analysis of massively parallel reporter assays enables prediction of regulatory function across cell types“. Human Mutation 40, Nr. 9 (September 2019): 1299–313. http://dx.doi.org/10.1002/humu.23820.

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Poon, Kok-Siong, Lily Chiu und Karen Mei-Ling Tan. „Laboratory Verification of a BRCA1 and BRCA2 Massively Parallel Sequencing Assay from Wet Bench to Bioinformatics for Germline DNA Analysis“. Global Medical Genetics 08, Nr. 02 (16.03.2021): 062–68. http://dx.doi.org/10.1055/s-0041-1726338.

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Abstract Introduction A robust genetic test for BRCA1 and BRCA2 genes is necessary for the diagnosis, prognosis, and treatment of patients with hereditary breast and ovarian cancer. We evaluated a commercial amplicon-based massively parallel sequencing (MPS) assay, BRCA MASTR Plus on the MiSeq platform, for germline BRCA genetic testing. Methods This study was performed on 31 DNA from cell lines and proficiency testing samples to establish the accuracy of the assay. A reference cell line DNA, NA12878 was used to determine the reproducibility of the assay. Discordant MPS result was resolved orthogonally by the current gold-standard Sanger sequencing method. Results The analytical accuracy, sensitivity, and specificity for variant detection were 93.55, 92.86, and 100.00%, respectively. Both sequencing depth and variant allele frequencies were highly reproducible by comparing the NA12878 DNA tested in three separate runs. The single discordant result, later confirmed by Sanger sequencing was due to the inability of the MASTR Reporter software to identify a 40-bp deletion in BRCA1. Conclusion The BRCA MASTR Plus assay on the MiSeq platform is accurate and reproducible for germline BRCA genetic testing, making it suitable for use in a clinical diagnostic laboratory. However, Sanger sequencing may still serve as a confirmatory method to improve diagnostic capability of the MPS assay.

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Davis, Jessica E., Kimberly D. Insigne, Eric M. Jones, Quinn A. Hastings, W. Clifford Boldridge und Sriram Kosuri. „Dissection of c-AMP Response Element Architecture by Using Genomic and Episomal Massively Parallel Reporter Assays“. Cell Systems 11, Nr. 1 (Juli 2020): 75–85. http://dx.doi.org/10.1016/j.cels.2020.05.011.

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Nuytemans, Karen, Derek J. van Booven, Natalia K. Hofmann, Farid Rajabli, Anthony J. Griswold, Christopher D. Brown, Margaret A. Pericak-Vance und Jeffery M. Vance. „P2-143: USING MASSIVELY PARALLEL REPORTER ASSAYS TO IDENTIFY PROTECTIVE FUNCTIONAL VARIANTS IN THE APOE REGION“. Alzheimer's & Dementia 15 (Juli 2019): P628. http://dx.doi.org/10.1016/j.jalz.2019.06.2550.

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Movva, Rajiv, Peyton Greenside, Georgi K. Marinov, Surag Nair, Avanti Shrikumar und Anshul Kundaje. „Deciphering regulatory DNA sequences and noncoding genetic variants using neural network models of massively parallel reporter assays“. PLOS ONE 14, Nr. 6 (17.06.2019): e0218073. http://dx.doi.org/10.1371/journal.pone.0218073.

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Karollus, Alexander, Žiga Avsec und Julien Gagneur. „Predicting mean ribosome load for 5’UTR of any length using deep learning“. PLOS Computational Biology 17, Nr. 5 (10.05.2021): e1008982. http://dx.doi.org/10.1371/journal.pcbi.1008982.

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The 5’ untranslated region plays a key role in regulating mRNA translation and consequently protein abundance. Therefore, accurate modeling of 5’UTR regulatory sequences shall provide insights into translational control mechanisms and help interpret genetic variants. Recently, a model was trained on a massively parallel reporter assay to predict mean ribosome load (MRL)—a proxy for translation rate—directly from 5’UTR sequence with a high degree of accuracy. However, this model is restricted to sequence lengths investigated in the reporter assay and therefore cannot be applied to the majority of human sequences without a substantial loss of information. Here, we introduced frame pooling, a novel neural network operation that enabled the development of an MRL prediction model for 5’UTRs of any length. Our model shows state-of-the-art performance on fixed length randomized sequences, while offering better generalization performance on longer sequences and on a variety of translation-related genome-wide datasets. Variant interpretation is demonstrated on a 5’UTR variant of the gene HBB associated with beta-thalassemia. Frame pooling could find applications in other bioinformatics predictive tasks. Moreover, our model, released open source, could help pinpoint pathogenic genetic variants.

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White, Michael A. „Understanding how cis -regulatory function is encoded in DNA sequence using massively parallel reporter assays and designed sequences“. Genomics 106, Nr. 3 (September 2015): 165–70. http://dx.doi.org/10.1016/j.ygeno.2015.06.003.

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Liu, Ying, Takuma Irie, Tetsushi Yada und Yutaka Suzuki. „A new computational method to predict transcriptional activity of a DNA sequence from diverse datasets of massively parallel reporter assays“. Nucleic Acids Research 45, Nr. 13 (22.05.2017): e124-e124. http://dx.doi.org/10.1093/nar/gkx396.

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Castaldi, Peter J., Feng Guo, Dandi Qiao, Fei Du, Zun Zar Chi Naing, Yan Li, Betty Pham et al. „Identification of Functional Variants in the FAM13A Chronic Obstructive Pulmonary Disease Genome-Wide Association Study Locus by Massively Parallel Reporter Assays“. American Journal of Respiratory and Critical Care Medicine 199, Nr. 1 (Januar 2019): 52–61. http://dx.doi.org/10.1164/rccm.201802-0337oc.

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Catizone, Allison N., Gizem Karsli Uzunbas, Petra Celadova, Sylvia Kuang, Daniel Bose und Morgan A. Sammons. „Locally acting transcription factors regulate p53-dependent cis-regulatory element activity“. Nucleic Acids Research 48, Nr. 8 (05.03.2020): 4195–213. http://dx.doi.org/10.1093/nar/gkaa147.

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Abstract The master tumor suppressor p53 controls transcription of a wide-ranging gene network involved in apoptosis, cell cycle arrest, DNA damage repair, and senescence. Recent studies revealed pervasive binding of p53 to cis-regulatory elements (CREs), which are non-coding segments of DNA that spatially and temporally control transcription through the combinatorial binding of local transcription factors. Although the role of p53 as a strong trans-activator of gene expression is well known, the co-regulatory factors and local sequences acting at p53-bound CREs are comparatively understudied. We designed and executed a massively parallel reporter assay (MPRA) to investigate the effect of transcription factor binding motifs and local sequence context on p53-bound CRE activity. Our data indicate that p53-bound CREs are both positively and negatively affected by alterations in local sequence context and changes to co-regulatory TF motifs. Our data suggest p53 has the flexibility to cooperate with a variety of transcription factors in order to regulate CRE activity. By utilizing different sets of co-factors across CREs, we hypothesize that global p53 activity is guarded against loss of any one regulatory partner, allowing for dynamic and redundant control of p53-mediated transcription.

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Santiago-Algarra, David, Lan T. M. Dao, Lydie Pradel, Alexandre España und Salvatore Spicuglia. „Recent advances in high-throughput approaches to dissect enhancer function“. F1000Research 6 (19.06.2017): 939. http://dx.doi.org/10.12688/f1000research.11581.1.

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The regulation of gene transcription in higher eukaryotes is accomplished through the involvement of transcription start site (TSS)-proximal (promoters) and -distal (enhancers) regulatory elements. It is now well acknowledged that enhancer elements play an essential role during development and cell differentiation, while genetic alterations in these elements are a major cause of human disease. Many strategies have been developed to identify and characterize enhancers. Here, we discuss recent advances in high-throughput approaches to assess enhancer activity, from the well-established massively parallel reporter assays to the recent clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based technologies. We highlight how these approaches contribute toward a better understanding of enhancer function, eventually leading to the discovery of new types of regulatory sequences, and how the alteration of enhancers can affect transcriptional regulation.

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Ferreira, Leonardo M. R., Torsten B. Meissner, Tarjei S. Mikkelsen, William Mallard, Charles W. O’Donnell, Tamara Tilburgs, Hannah A. B. Gomes et al. „A distant trophoblast-specific enhancer controls HLA-G expression at the maternal–fetal interface“. Proceedings of the National Academy of Sciences 113, Nr. 19 (13.04.2016): 5364–69. http://dx.doi.org/10.1073/pnas.1602886113.

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HLA-G, a nonclassical HLA molecule uniquely expressed in the placenta, is a central component of fetus-induced immune tolerance during pregnancy. The tissue-specific expression of HLA-G, however, remains poorly understood. Here, systematic interrogation of the HLA-G locus using massively parallel reporter assay (MPRA) uncovered a previously unidentified cis-regulatory element 12 kb upstream of HLA-G with enhancer activity, Enhancer L. Strikingly, clustered regularly-interspaced short palindromic repeats (CRISPR)/Cas9-mediated deletion of this enhancer resulted in ablation of HLA-G expression in JEG3 cells and in primary human trophoblasts isolated from placenta. RNA-seq analysis demonstrated that Enhancer L specifically controls HLA-G expression. Moreover, DNase-seq and chromatin conformation capture (3C) defined Enhancer L as a cell type-specific enhancer that loops into the HLA-G promoter. Interestingly, MPRA-based saturation mutagenesis of Enhancer L identified motifs for transcription factors of the CEBP and GATA families essential for placentation. These factors associate with Enhancer L and regulate HLA-G expression. Our findings identify long-range chromatin looping mediated by core trophoblast transcription factors as the mechanism controlling tissue-specific HLA-G expression at the maternal–fetal interface. More broadly, these results establish the combination of MPRA and CRISPR/Cas9 deletion as a powerful strategy to investigate human immune gene regulation.

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Ainsworth, Hannah C., Timothy D. Howard und Carl D. Langefeld. „Intrinsic DNA topology as a prioritization metric in genomic fine-mapping studies“. Nucleic Acids Research 48, Nr. 20 (21.10.2020): 11304–21. http://dx.doi.org/10.1093/nar/gkaa877.

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Abstract In genomic fine-mapping studies, some approaches leverage annotation data to prioritize likely functional polymorphisms. However, existing annotation resources can present challenges as many lack information for novel variants and/or may be uninformative for non-coding regions. We propose a novel annotation source, sequence-dependent DNA topology, as a prioritization metric for fine-mapping. DNA topology and function are well-intertwined, and as an intrinsic DNA property, it is readily applicable to any genomic region. Here, we constructed and applied Minor Groove Width (MGW) as a prioritization metric. Using an established MGW-prediction method, we generated a MGW census for 199 038 197 SNPs across the human genome. Summarizing a SNP’s change in MGW (ΔMGW) as a Euclidean distance, ΔMGW exhibited a strongly right-skewed distribution, highlighting the infrequency of SNPs that generate dissimilar shape profiles. We hypothesized that phenotypically-associated SNPs can be prioritized by ΔMGW. We tested this hypothesis in 116 regions analyzed by a Massively Parallel Reporter Assay and observed enrichment of large ΔMGW for functional polymorphisms (P = 0.0007). To illustrate application in fine-mapping studies, we applied our MGW-prioritization approach to three non-coding regions associated with systemic lupus erythematosus. Together, this study presents the first usage of sequence-dependent DNA topology as a prioritization metric in genomic association studies.

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Shinozuka, Hiroshi, und John W. Forster. „Use of the melting curve assay as a means for high-throughput quantification of Illumina sequencing libraries“. PeerJ 4 (04.08.2016): e2281. http://dx.doi.org/10.7717/peerj.2281.

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Background.Multiplexed sequencing is commonly performed on massively parallel short-read sequencing platforms such as Illumina, and the efficiency of library normalisation can affect the quality of the output dataset. Although several library normalisation approaches have been established, none are ideal for highly multiplexed sequencing due to issues of cost and/or processing time.Methods.An inexpensive and high-throughput library quantification method has been developed, based on an adaptation of the melting curve assay. Sequencing libraries were subjected to the assay using the Bio-Rad Laboratories CFX ConnectTMReal-Time PCR Detection System. The library quantity was calculated through summation of reduction of relative fluorescence units between 86 and 95 °C.Results.PCR-enriched sequencing libraries are suitable for this quantification without pre-purification of DNA. Short DNA molecules, which ideally should be eliminated from the library for subsequent processing, were differentiated from the target DNA in a mixture on the basis of differences in melting temperature. Quantification results for long sequences targeted using the melting curve assay were correlated with those from existing methods (R2> 0.77), and that observed from MiSeq sequencing (R2= 0.82).Discussion.The results of multiplexed sequencing suggested that the normalisation performance of the described method is equivalent to that of another recently reported high-throughput bead-based method, BeNUS. However, costs for the melting curve assay are considerably lower and processing times shorter than those of other existing methods, suggesting greater suitability for highly multiplexed sequencing applications.

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Belliveau, Nathan M., Stephanie L. Barnes, William T. Ireland, Daniel L. Jones, Michael J. Sweredoski, Annie Moradian, Sonja Hess, Justin B. Kinney und Rob Phillips. „Systematic approach for dissecting the molecular mechanisms of transcriptional regulation in bacteria“. Proceedings of the National Academy of Sciences 115, Nr. 21 (04.05.2018): E4796—E4805. http://dx.doi.org/10.1073/pnas.1722055115.

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Gene regulation is one of the most ubiquitous processes in biology. However, while the catalog of bacterial genomes continues to expand rapidly, we remain ignorant about how almost all of the genes in these genomes are regulated. At present, characterizing the molecular mechanisms by which individual regulatory sequences operate requires focused efforts using low-throughput methods. Here, we take a first step toward multipromoter dissection and show how a combination of massively parallel reporter assays, mass spectrometry, and information-theoretic modeling can be used to dissect multiple bacterial promoters in a systematic way. We show this approach on both well-studied and previously uncharacterized promoters in the enteric bacterium Escherichia coli. In all cases, we recover nucleotide-resolution models of promoter mechanism. For some promoters, including previously unannotated ones, the approach allowed us to further extract quantitative biophysical models describing input–output relationships. Given the generality of the approach presented here, it opens up the possibility of quantitatively dissecting the mechanisms of promoter function in E. coli and a wide range of other bacteria.

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Örd, Tiit, Kadri Õunap, Lindsey K. Stolze, Redouane Aherrahrou, Valtteri Nurminen, Anu Toropainen, Ilakya Selvarajan et al. „Single-Cell Epigenomics and Functional Fine-Mapping of Atherosclerosis GWAS Loci“. Circulation Research 129, Nr. 2 (09.07.2021): 240–58. http://dx.doi.org/10.1161/circresaha.121.318971.

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Rationale: Genome-wide association studies have identified hundreds of loci associated with coronary artery disease (CAD). Many of these loci are enriched in cisregulatory elements but not linked to cardiometabolic risk factors nor to candidate causal genes, complicating their functional interpretation. Objective: Single-nucleus chromatin accessibility profiling of the human atherosclerotic lesions was used to investigate cell type–specific patterns of cisregulatory elements, to understand transcription factors establishing cell identity, and to interpret CAD-relevant, noncoding genetic variation. Methods and Results: We used single-nucleus ATAC-seq (assay for transposase-accessible chromatin with sequencing) to generate DNA accessibility maps in >7000 cells derived from human atherosclerotic lesions. We identified 5 major lesional cell types including endothelial cells, smooth muscle cells, monocyte/macrophages, natural killer/T cells, and B cells and further investigated subtype characteristics of macrophages and smooth muscle cells transitioning into fibromyocytes. We demonstrated that CAD-associated genetic variants are particularly enriched in endothelial and smooth muscle cell–specific open chromatin. Using single-cell coaccessibility and cis–expression quantitative trait loci information, we prioritized putative target genes and candidate regulatory elements for ≈30% of all known CAD loci. Finally, we performed genome-wide experimental fine-mapping of the CAD variants identified in genome-wide association studies using epigenetic quantitative trait loci analysis in primary human aortic endothelial cells and self-transcribing active regulatory region sequencing (STARR-Seq) massively parallel reporter assay in smooth muscle cells. This analysis identified potential causal single-nucleotide polymorphisms (SNPs) and the associated target gene for over 30 CAD loci. We present several examples where the chromatin accessibility and gene expression could be assigned to one cell type predicting the cell type of action for CAD loci. Conclusions: These findings highlight the potential of applying single-nucleus ATAC-seq to human tissues in revealing relative contributions of distinct cell types to diseases and in identifying genes likely to be influenced by noncoding genome-wide association study variants.

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Grossman, Sharon R., Xiaolan Zhang, Li Wang, Jesse Engreitz, Alexandre Melnikov, Peter Rogov, Ryan Tewhey et al. „Systematic dissection of genomic features determining transcription factor binding and enhancer function“. Proceedings of the National Academy of Sciences 114, Nr. 7 (30.01.2017): E1291—E1300. http://dx.doi.org/10.1073/pnas.1621150114.

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Enhancers regulate gene expression through the binding of sequence-specific transcription factors (TFs) to cognate motifs. Various features influence TF binding and enhancer function—including the chromatin state of the genomic locus, the affinities of the binding site, the activity of the bound TFs, and interactions among TFs. However, the precise nature and relative contributions of these features remain unclear. Here, we used massively parallel reporter assays (MPRAs) involving 32,115 natural and synthetic enhancers, together with high-throughput in vivo binding assays, to systematically dissect the contribution of each of these features to the binding and activity of genomic regulatory elements that contain motifs for PPARγ, a TF that serves as a key regulator of adipogenesis. We show that distinct sets of features govern PPARγ binding vs. enhancer activity. PPARγ binding is largely governed by the affinity of the specific motif site and higher-order features of the larger genomic locus, such as chromatin accessibility. In contrast, the enhancer activity of PPARγ binding sites depends on varying contributions from dozens of TFs in the immediate vicinity, including interactions between combinations of these TFs. Different pairs of motifs follow different interaction rules, including subadditive, additive, and superadditive interactions among specific classes of TFs, with both spatially constrained and flexible grammars. Our results provide a paradigm for the systematic characterization of the genomic features underlying regulatory elements, applicable to the design of synthetic regulatory elements or the interpretation of human genetic variation.

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Findley, Anthony S., Xinjun Zhang, Carly Boye, Yen Lung Lin, Cynthia A. Kalita, Luis Barreiro, Kirk E. Lohmueller, Roger Pique-Regi und Francesca Luca. „A signature of Neanderthal introgression on molecular mechanisms of environmental responses“. PLOS Genetics 17, Nr. 9 (27.09.2021): e1009493. http://dx.doi.org/10.1371/journal.pgen.1009493.

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Ancient human migrations led to the settlement of population groups in varied environmental contexts worldwide. The extent to which adaptation to local environments has shaped human genetic diversity is a longstanding question in human evolution. Recent studies have suggested that introgression of archaic alleles in the genome of modern humans may have contributed to adaptation to environmental pressures such as pathogen exposure. Functional genomic studies have demonstrated that variation in gene expression across individuals and in response to environmental perturbations is a main mechanism underlying complex trait variation. We considered gene expression response to in vitro treatments as a molecular phenotype to identify genes and regulatory variants that may have played an important role in adaptations to local environments. We investigated if Neanderthal introgression in the human genome may contribute to the transcriptional response to environmental perturbations. To this end we used eQTLs for genes differentially expressed in a panel of 52 cellular environments, resulting from 5 cell types and 26 treatments, including hormones, vitamins, drugs, and environmental contaminants. We found that SNPs with introgressed Neanderthal alleles (N-SNPs) disrupt binding of transcription factors important for environmental responses, including ionizing radiation and hypoxia, and for glucose metabolism. We identified an enrichment for N-SNPs among eQTLs for genes differentially expressed in response to 8 treatments, including glucocorticoids, caffeine, and vitamin D. Using Massively Parallel Reporter Assays (MPRA) data, we validated the regulatory function of 21 introgressed Neanderthal variants in the human genome, corresponding to 8 eQTLs regulating 15 genes that respond to environmental perturbations. These findings expand the set of environments where archaic introgression may have contributed to adaptations to local environments in modern humans and provide experimental validation for the regulatory function of introgressed variants.

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Romanov, S. E., D. A. Kalashnikova und P. P. Laktionov. „Methods of massive parallel reporter assays for investigation of enhancers“. Vavilov Journal of Genetics and Breeding 25, Nr. 3 (02.06.2021): 344–55. http://dx.doi.org/10.18699/vj21.038.

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The correct deployment of genetic programs for development and differentiation relies on finely coordinated regulation of specific gene sets. Genomic regulatory elements play an exceptional role in this process. There are few types of gene regulatory elements, including promoters, enhancers, insulators and silencers. Alterations of gene regulatory elements may cause various pathologies, including cancer, congenital disorders and autoimmune diseases. The development of high-throughput genomic assays has made it possible to significantly accelerate the accumulation of information about the characteristic epigenetic properties of regulatory elements. In combination with high-throughput studies focused on the genome-wide distribution of epigenetic marks, regulatory proteins and the spatial structure of chromatin, this significantly expands the understanding of the principles of epigenetic regulation of genes and allows potential regulatory elements to be searched for in silico. However, common experimental approaches used to study the local characteristics of chromatin have a number of technical limitations that may reduce the reliability of computational identification of genomic regulatory sequences. Taking into account the variability of the functions of epigenetic determinants and complex multicomponent regulation of genomic elements activity, their functional verification is often required. A plethora of methods have been developed to study the functional role of regulatory elements on the genome scale. Common experimental approaches for in silico identification of regulatory elements and their inherent technical limitations will be described. The present review is focused on original high-throughput methods of enhancer activity reporter analysis that are currently used to validate predicted regulatory elements and to perform de novo searches. The methods described allow assessing the functional role of the nucleotide sequence of a regulatory element, to determine its exact boundaries and to assess the influence of the local state of chromatin on the activity of enhancers and gene expression. These approaches have contributed substantially to the understanding of the fundamental principles of gene regulation.

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Kohlmann, Alexander, Andreas Roller, Andreia Albuquerque, Sabrina Kuznia, Sandra Weissmann, Sabine Jeromin, Wolfgang Kern, Claudia Haferlach, Susanne Schnittger und Torsten Haferlach. „A 13-Gene Panel Targeted To Investigate CLL By Next-Generation Amplicon Deep-Sequencing Can Be Successfully Implemented In Routine Diagnostics“. Blood 122, Nr. 21 (15.11.2013): 867. http://dx.doi.org/10.1182/blood.v122.21.867.867.

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Abstract Introduction Massively parallel next-generation sequencing (NGS) data have changed the landscape of molecular mutations in chronic lymphocytic leukemia (CLL). The number of molecular markers continues to constantly increase. As such, physicians and laboratories face a great unmet yet challenging need to test panels of genes at a high level of sensitivity. Aim To develop an assay that is easily adoptable to adjust gene targets and amplicons according to current state-of-the-art evidence regarding the published landscape of mutations in CLL. Methods We developed a sensitive deep-sequencing assay for routine diagnostics. In total, 13 genes with relevance in CLL providing in part adverse prognostic information were chosen: ATM, BIRC3, BRAF (V600), FBXW7, KLHL6, KRAS, NOTCH1 (PEST domain), NRAS, MYD88, POT1, SF3B1 (HEAT repeats), TP53, and XPO1. Targets of interest comprised either complete coding gene regions or hotspots. In summary, 323 amplicons were designed with a median length of 204 bp (range 150-240 bp), representing a total target sequence of 39.36 kb. The sequencing library was constructed starting off 2.2 μg genomic DNA per patient using a single-plex microdroplet-based assay (RainDance, Lexington, MA). Sequencing data was generated using the MiSeq instrument (Illumina, San Diego, CA) loading up to 10 patients per run. The total turn-around time of the assay was less than 5 days. As a proof-of-principle cohort, 18 clinically well-annotated CLL patients were analyzed during the evaluation phase. The median age was 78 years (range: 52 – 87 years). Results Using the 500 cycles sequencing by-synthesis chemistry, in median 7,262 millions of paired-end reads were generated per run. This resulted in a median coverage per gene of 7,476 reads (range: 5,595 - 10,337). (1) In this cohort of 18 cases, a total of 71 mutation analyses had already been previously performed for eight of the 13 genes using either capillary Sanger sequencing or alternative amplicon deep-sequencing assays (454 LifeSciences or Illumina MiSeq). In detail, in these 8 genes these 71 assays detected 56 known polymorphisms or mutations in ATM (n=8), BIRC3 (n=6), FBXW7 (n=4), MYD88 (n=4), NOTCH1 (n=10), SF3B1 (n=5), TP53 (n=14), and XPO1 (n=4) and 28 analyses revealed a wild-type status. When comparing these results with data obtained using the 13-gene NGS panel, in all 84/84 (100%) parallel assessments concordant results were obtained underlining the robustness of this assay. (2) Overall and extending the previous results, the comprehensive 13-gene NGS panel then detected in 18/18 patients a total of 46 mutations in 10 of the 13 genes with a range of 1-5 mutations per case (median: 2). The mutation types comprised 22 missense, 4 nonsense, 16 frame-shifts, 3 insertions and 1 splice-site alterations. In median, the coverage per variant was 10,390-fold, thus enabling a sensitive detection of mutations at a lower limit of detection set at 3%. The mutation burden ranged from 3.0% to 62.0%. 18/46 (39.13%) mutations were detected with a clone size <20%, thus being detected only due to the higher sensitivity of this assay in comparison to direct capillary Sanger sequencing. With respect to the technical limit of detecting larger alterations, a 34 bp deletion variant (NOTCH1; c.7403_7436del) was successfully sequenced. Moreover, a common theme in hematological malignancies is the emergence of novel prognostic scoring systems, integrating molecular mutations and cytogenetic lesions into revised survival prediction models. Importantly, a number of patients (14/18) was detected to harbor mutations in genes reported to be associated with decreased overall survival, both in high-risk (e.g. TP53, BIRC3) and intermediate-risk (NOTCH1, SF3B1) categories according to Rossi et al., 2013 (Blood;121:1403-12). As such, detecting these adverse somatic alterations may influence the course of therapy for these patients underlining the utility of such a screening panel. Conclusion We demonstrated that microdroplet-based sample preparation enabled to robustly target 13 genes for next-generation sequencing in a routine diagnostics environment. This included also larger gene targets such as ATM, being represented by 119 amplicons. Thus, this approach provides the potential to screen for prognostically relevant mutations in all CLL patients in a fast and comprehensive way providing actionable information suitable to guide therapy. Disclosures: Kohlmann MLL Munich Leukemia Laboratory: Employment. Roller:MLL Munich Leukemia Laboratory: Employment. Albuquerque:MLL Munich Leukemia Laboratory: Employment. Kuznia:MLL Munich Leukemia Laboratory: Employment. Weissmann:MLL Munich Leukemia Laboratory: Employment. Jeromin:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Steiner, Laurie A., Vincent Schulz, Yelena Maksimova, Nancy E. Seidel, David M. Bodine und Patrick G. Gallagher. „Unbiased Identification of Functional Barrier Insulators in Primary Human Erythroid Cells“,. Blood 118, Nr. 21 (18.11.2011): 3385. http://dx.doi.org/10.1182/blood.v118.21.3385.3385.

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Abstract Abstract 3385 Barrier insulators function to actively maintain the boundaries between heterochromatin and euchromatin. They are critical for regulation of cell-type specific gene expression in normal development and differentiation. Mutations that disrupt barrier insulator function have been associated with developmental disorders, malignancies, and inherited hemolytic anemias. Barrier insulators are poorly understood in mammalian cells, with much of the available data coming from model organisms. In vertebrates, the best characterized barrier insulator is the 5' hypersensitive site in the LCR of the chicken β-globin gene cluster (cHS4). In cHS4, barrier insulator function is mediated by binding of the upstream stimulatory factor (USF) proteins, which bind specific DNA sequences and recruit multiple regulatory proteins, including histone aceytltranserases (HATs) and histone methyltransferases (MTs), which maintain DNA in a euchromatin state. The cHS4 barrier also recruits the protein VEZF1, recently shown to mediate protection of DNA from methylation. We hypothesize that there is a common regulatory signature for cell-type specific barrier insulators characterized by binding of the USF proteins, with recruitment of HATs, MTs, and other proteins in the genome of human erythroid cells. To test this hypothesis, we utilized chromatin immunoprecipitation coupled with massively parallel sequencing (ChIP-seq) to generate genome-wide maps of barrier-associated proteins and histone modifications in primary human erythroid cells (R3/R4 stage). Regions where barrier-associated proteins co-localize, representing potential barrier insulators, were identified then subjected to functional analysis in position effect variegation (PEV) assays. Genome-wide, 3825 sites bound the USF proteins USF1 and USF2 with their associated MTs (PRMT1/PRMT4), and HATs (P300, PCAF, SRC1). The genome-wide binding of VEZF1 was compared to the binding of the USFs, MTs, and HATs. VEZF1 bound 1129 (30%) of the potential barrier sites. The role of CTCF in barrier insulators is controversial. It is dispensable for cHS4 barrier function in chicken erythroid cells, but in human cells, it marks chromatin boundaries in a cell-type specific manner. CTCF ChIP-seq in erythroid cells revealed that a very large number of the barrier-associated sites (3382, 88%) bound CTCF. Together, 1167 sites bound all 9 factors. These sites were located primarily in gene promoters (42%) and 5' untranslated regions (23%), consistent with data from Drosophila, where barriers are frequently associated with gene promoters. Active barriers are associated with an “open” chromatin structure and lack CpG methylation, thus these epigenetic marks were assessed at the predicted barrier sites. The majority of sites, 96%, had the active histone mark H3K4me2, while only 0.02% were positive for the repressive histone mark H3K27me3. To assess CpG methylation, methyl binding domain pull down was coupled with massively parallel sequencing (MethylSeq). 3676 regions of CpG methylation were identified, but none overlapped with the barrier signature. PEV assays, which assesses the ability of a region of DNA to protect a reporter gene from heterochromatin-mediated silencing, were used to determine if selected sites identified by ChIP-seq studies had barrier insulator function in vivo. Constructs containing an EF1alpha promoter directing an EGFP reporter gene-IRES-hygromycin cassette were flanked by potential barriers and stably transfected into K562 cells. Results from single copy clones were normalized to the cHS4 positive control. Sites tested included an intergenic site on chromosome 11 located >100kb from any known gene (site 1), which bound the USFs, PRMTs, PCAF, SRC1, and CTCF, and a site in intron 1 of the band 3 gene (site 2), which bound the USFs, PRMT4, P300, PCAF, and SRC1. Both sites were shown to have barrier activity (site 1 x2= 6.77, p<0.01 and site 2 x2= 3.30, p<0.06), demonstrating that our molecular signature can predict functional barrier insulators. The orientation dependence of vertebrate barrier elements has never been described. When site 1 and 2 were analyzed in the opposite orientation relative to the direction of transcription, neither had barrier function. Unbiased identification of barrier insulators on a genome wide scale will provide novel insights into normal erythropoiesis and its perturbation in human disease. Disclosures: No relevant conflicts of interest to declare.

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Cai, Wei, Yu-Jui Chiu, Valya Ramakrishnan, Yihuan Tsai, Clark Chen und Yu-Hwa Lo. „A single-cell translocation and secretion assay (TransSeA)“. Lab on a Chip 18, Nr. 20 (2018): 3154–62. http://dx.doi.org/10.1039/c8lc00821c.

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Roberts, Brian S., E. Christopher Partridge, Bryan A. Moyers, Vikram Agarwal, Kimberly M. Newberry, Beth K. Martin, Jay Shendure, Richard M. Myers und Gregory M. Cooper. „Genome-wide strand asymmetry in massively parallel reporter activity favors genic strands“. Genome Research 31, Nr. 5 (20.04.2021): 866–76. http://dx.doi.org/10.1101/gr.270751.120.

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Oldoni, Fabio, Drew Bader, Chiara Fantinato, Sharon C. Wootton, Robert Lagacé, Ryo Hasegawa, Joseph Chang, Kenneth Kidd und Daniele Podini. „A massively parallel sequencing assay of microhaplotypes for mixture deconvolution“. Forensic Science International: Genetics Supplement Series 7, Nr. 1 (Dezember 2019): 522–24. http://dx.doi.org/10.1016/j.fsigss.2019.10.075.

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Fellmann, Christof, Johannes Zuber, Katherine McJunkin, Kenneth Chang, Colin D. Malone, Ross A. Dickins, Qikai Xu et al. „Functional Identification of Optimized RNAi Triggers Using a Massively Parallel Sensor Assay“. Molecular Cell 41, Nr. 6 (März 2011): 733–46. http://dx.doi.org/10.1016/j.molcel.2011.02.008.

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Hong, Yuanyuan, Weizhi Chen, Huiting Yan, Linlin Yan, Xuexia Zeng und Yipeng Song. „Validation of a comprehensive cancer genomic profiling assay based on massively parallel DNA sequencing.“ Journal of Clinical Oncology 37, Nr. 15_suppl (20.05.2019): e13138-e13138. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.e13138.

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e13138 Background: Validation is very important for diagnostic assays, here we describe a validation process of a comprehensive cancer genomic profiling assay based on massively parallel DNA sequencing. This assay can detect base substitutions, short insertions and deletions, copy number alterations, tumor mutation burden (TMB) and microsatellite instability (MSI) across 543 cancer-related genes from tumor specimens or plasma. Methods: Analytical validation was conducted with 20 cell line pools whose mutation were verified by digital droplet PCR (ddPCR). Clinical validation was conducted with Proficiency Test samples and clinical samples whose mutation were verified by ddPCR. Sensitivity, positive predictive value (PPV) and precision of tumor specimens and plasma were assessed across the reportable range of the assay. For measurement of TMB， NGS libraries of a cohort of tumor samples were tested by whole exosome panel (WES) and this assay respectively. And clinical tumor tissue samples whose MSI status were identified by fluorescent PCR-capillary electrophoresis were also tested by this assay. Results: The SNV/Indel LOD95 was 0.6% for hot-spot variants, and 1.3% for panel-wised variants of tumor tissue samples with high specificity (PPV >99%). The SNV/Indel LOD95 was 0.4% for hot-spot variants, and 1.1% for panel-wised variants of plasma samples (cfDNA≥15ng), with PPV >99%. LOD95 of CNV was 2.2-2.3, with PPV>99%. Precision of CNV was higher than 95%, precision of SNV/Indel was higher than 97%. Concordance between TMB results tested by WES and this assay was 0.94. MSI results identified by this assay was the same as fluorescent PCR-capillary electrophoresis method. Conclusions: In summary, we present the analytical and clinical validation of a comprehensive NGS-based diagnostic assay for comprehensive tumor genomic profiling.

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