Positive predictive values could be increased by factors of 1.07 to 1.27 in case of single nucleotide variant calling and by factors of 3.33 to 53.87 in case of indel call- ing.Show all 5 authors Hide Download full-text PDF Read full-text Download full-text PDF Read full-text Download citation Copy link Link copied Read full-text Download citation Copy link Link copied Citations (6) References (36) Figures (4) Abstract and Figures Background.There are various next-generation sequencing techniques, all of them striving to replace Sanger sequencing as the gold standard.However, false positive calls of single nucleotide variants and especially indels are a widely known problem of basically all sequencing platforms.
Contemporary Strategy Analysis Grant Torrent Torrent PGM AndWe considered three common next-generation sequencersRoche 454, Ion Torrent PGM and Illumina NextSeqand applied standard as well as optimized variant calling pipelines. Optimization was achieved by combining information of 23 diverse parameters characterizing the reported variants and generating individually calibrated generalized linear models. Models were calibrated using amplicon-based targeted sequencing data (19 genes, 28,775 bp) from seven to 12 myelodysplastic syndrome patients. Evaluation of the optimized pipelines and platforms was performed using sequencing data from three additional myelodysplastic syndrome patients. ![]() Analysis of the parameters characterizing the true and false positive calls revealed significant platform- and variant specific differences. Application of optimized variant calling pipelines considerably improved results. Positive predictive values could be increased by factors of 1.07 to 1.27 in case of single nucleotide variant calling and by factors of 3.33 to 53.87 in case of indel calling. Application of the optimized variant calling pipelines leads to comparable results for all next-generation sequencing platforms analyzed. However, regarding clinical diagnostics it needs to be considered that even the optimized results still contained false positive as well as false negative calls. Overview of the variant calling pipeline (steps marked by dashed frames are only performed in case of the variant calling pipeline with GLM).. Overview of the parameters investigated for the variant calling pipeline with GLM. Median coverage of the genes in the intersecting target region in the case of 454 (black), Ion Torrent (red) and Illumina (green) considering the comparison data set. Normalized relative variable importance for all parameters characterizing SNVs, considering 454, Ion Torrent and Illumina NextSeq sequencing data. Figures - available via license: Creative Commons Attribution 4.0 International Content may be subject to copyright. Contemporary Strategy Analysis Grant Torrent For Free Public FullDiscover the worlds research 20 million members 135 million publications 700k research projects Join for free Public Full-text 1 Available via license: CC BY 4.0 Content may be subject to copyright. Methods We considered three common next-generation sequencersRoche 454, Ion Torrent PGM and Illumina NextSeqand applied standard as well as optimized variant calling pipelines. ![]() Evaluation of the optimized pipe- lines and platforms was performed using sequencing data from three additional myelodys- plastic syndrome patients. Results Using standard analysis methods, true mutations were missed and the obtained results con- tained many artifactsno matter which platform was considered. Analysis of the parame- ters characterizing the true and false positive calls revealed significant platform- and variant specific differences.
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