title: Reproducibility of Variant Calls in Replicate Next Generation Sequencing Experiments.
abstract: Nucleotide alterations detected by next generation sequencing are not always true biological changes but could represent sequencing errors. Even highly accurate methods can yield substantial error rates when applied to millions of nucleotides. In this study, we examined the reproducibility of nucleotide variant calls in replicate sequencing experiments of the same genomic DNA. We performed targeted sequencing of all known human protein kinase genes (kinome) (~3.2 Mb) using the SOLiD v4 platform. Seventeen breast cancer samples were sequenced in duplicate (n=14) or triplicate (n=3) to assess concordance of all calls and single nucleotide variant (SNV) calls. The concordance rates over the entire sequenced region were >99.99%, while the concordance rates for SNVs were 54.3-75.5%. There was substantial variation in basic sequencing metrics from experiment to experiment. The type of nucleotide substitution and genomic location of the variant had little impact on concordance but concordance increased with coverage level, variant allele count (VAC), variant allele frequency (VAF), variant allele quality and p-value of SNV-call. The most important determinants of concordance were VAC and VAF. Even using the highest stringency of QC metrics the reproducibility of SNV calls was around 80% suggesting that erroneous variant calling can be as high as 20-40% in a single experiment. The sequence data have been deposited into the European Genome-phenome Archive (EGA) with accession number EGAS00001000826.
authors.affiliation: Departments of Bioinformatics and Computational Biology, University of Texas MD Anderson Cancer Center, Houston, Texas, United States of America.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Qi
authors.affiliation: Department of Experimental Therapeutics, University of Texas MD Anderson Cancer Center, Houston, Texas, United States of America.
authors.forename: Xiuping
authors.initials: X
authors.lastname: Liu
authors.affiliation: Department of Experimental Therapeutics, University of Texas MD Anderson Cancer Center, Houston, Texas, United States of America.
authors.forename: Chang-Gong
authors.initials: CG
authors.lastname: Liu
authors.affiliation: Department of Breast Medical Oncology, University of Texas MD Anderson Cancer Center, Houston, Texas, United States of America.
authors.forename: Bailing
authors.initials: B
authors.lastname: Wang
authors.affiliation: Department of Biostatistics, University of Texas MD Anderson Cancer Center, Houston, Texas, United States of America.
authors.forename: Kenneth R
authors.initials: KR
authors.lastname: Hess
authors.affiliation: Department of Pathology, University of Texas MD Anderson Cancer Center, Houston, Texas, United States of America.
authors.forename: W Fraser
authors.initials: WF
authors.lastname: Symmans
authors.affiliation: Yale Cancer Center, Yale School of Medicine, New Haven, Connecticut, United States of America.
authors.forename: Weiwei
authors.initials: W
authors.lastname: Shi
authors.affiliation: Yale Cancer Center, Yale School of Medicine, New Haven, Connecticut, United States of America.
authors.forename: Lajos
authors.initials: L
authors.lastname: Pusztai
mesh_terms: D000483:Alleles; D016477:Artifacts; D001943:Breast Neoplasms / Q000235:genetics* / Q000473:pathology; D004247:DNA / Q000235:genetics; D005260:Female; D005787:Gene Frequency; D015894:Genome, Human; D059014:High-Throughput Nucleotide Sequencing / Q000706:statistics & numerical data*; D006801:Humans; D020641:Polymorphism, Single Nucleotide; D011494:Protein Kinases / Q000235:genetics*; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000706:statistics & numerical data*
chemical_list: D004247:DNA; D011494:Protein Kinases