title: From next-generation sequencing alignments to accurate comparison and validation of single-nucleotide variants: the pibase software.
abstract: Scientists working with single-nucleotide variants (SNVs), inferred by next-generation sequencing software, often need further information regarding true variants, artifacts and sequence coverage gaps. In clinical diagnostics, e.g. SNVs must usually be validated by visual inspection or several independent SNV-callers. We here demonstrate that 0.5-60% of relevant SNVs might not be detected due to coverage gaps, or might be misidentified. Even low error rates can overwhelm the true biological signal, especially in clinical diagnostics, in research comparing healthy with affected cells, in archaeogenetic dating or in forensics. For these reasons, we have developed a package called pibase, which is applicable to diploid and haploid genome, exome or targeted enrichment data. pibase extracts details on nucleotides from alignment files at user-specified coordinates and identifies reproducible genotypes, if present. In test cases pibase identifies genotypes at 99.98% specificity, 10-fold better than other tools. pibase also provides pair-wise comparisons between healthy and affected cells using nucleotide signals (10-fold more accurately than a genotype-based approach, as we show in our case study of monozygotic twins). This comparison tool also solves the problem of detecting allelic imbalance within heterozygous SNVs in copy number variation loci, or in heterogeneous tumor sequences.
authors.affiliation: Institute of Clinical Molecular Biology, Christian-Albrechts-University Kiel, D-24105 Kiel, Germany. m.forster@ikmb.uni-kiel.de
authors.forename: Michael
authors.initials: M
authors.lastname: Forster
authors.forename: Peter
authors.initials: P
authors.lastname: Forster
authors.forename: Abdou
authors.initials: A
authors.lastname: Elsharawy
authors.forename: Georg
authors.initials: G
authors.lastname: Hemmrich
authors.forename: Benjamin
authors.initials: B
authors.lastname: Kreck
authors.forename: Michael
authors.initials: M
authors.lastname: Wittig
authors.forename: Ingo
authors.initials: I
authors.lastname: Thomsen
authors.forename: Björn
authors.initials: B
authors.lastname: Stade
authors.forename: Matthias
authors.initials: M
authors.lastname: Barann
authors.forename: David
authors.initials: D
authors.lastname: Ellinghaus
authors.forename: Britt-Sabina
authors.initials: BS
authors.lastname: Petersen
authors.forename: Sandra
authors.initials: S
authors.lastname: May
authors.forename: Espen
authors.initials: E
authors.lastname: Melum
authors.forename: Markus B
authors.initials: MB
authors.lastname: Schilhabel
authors.forename: Andreas
authors.initials: A
authors.lastname: Keller
authors.forename: Stefan
authors.initials: S
authors.lastname: Schreiber
authors.forename: Philip
authors.initials: P
authors.lastname: Rosenstiel
authors.forename: Andre
authors.initials: A
authors.lastname: Franke
mesh_terms: D014644:Genetic Variation; D023281:Genomics; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D010802:Phylogeny; D015203:Reproducibility of Results; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D012984:Software; D014430:Twins, Monozygotic / Q000235:genetics