title: Biased estimates of clonal evolution and subclonal heterogeneity can arise from PCR duplicates in deep sequencing experiments.
abstract: Accurate allele frequencies are important for measuring subclonal heterogeneity and clonal evolution. Deep-targeted sequencing data can contain PCR duplicates, inflating perceived read depth. Here we adapted the Illumina TruSeq Custom Amplicon kit to include single molecule tagging (SMT) and show that SMT-identified duplicates arise from PCR. We demonstrate that retention of PCR duplicate reads can imply clonal evolution when none exists, while their removal effectively controls the false positive rate. Additionally, PCR duplicates alter estimates of subclonal heterogeneity in tumor samples. Our method simplifies PCR duplicate identification and emphasizes their removal in studies of tumor heterogeneity and clonal evolution.
authors.forename: Erin N
authors.initials: EN
authors.lastname: Smith
authors.forename: Kristen
authors.initials: K
authors.lastname: Jepsen
authors.forename: Mahdieh
authors.initials: M
authors.lastname: Khosroheidari
authors.forename: Laura Z
authors.initials: LZ
authors.lastname: Rassenti
authors.forename: Matteo
authors.initials: M
authors.lastname: D'Antonio
authors.forename: Emanuela M
authors.initials: EM
authors.lastname: Ghia
authors.forename: Dennis A
authors.initials: DA
authors.lastname: Carson
authors.forename: Catriona Hm
authors.initials: CH
authors.lastname: Jamieson
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Kipps
authors.forename: Kelly A
authors.initials: KA
authors.lastname: Frazer
mesh_terms: D060965:Clonal Evolution; D005189:False Positive Reactions; D005787:Gene Frequency; D018740:Genetic Heterogeneity; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods; D012984:Software
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