title: Benefits and Challenges with Applying Unique Molecular Identifiers in Next Generation Sequencing to Detect Low Frequency Mutations.
abstract: Indexing individual template molecules with a unique identifier (UID) before PCR and deep sequencing is promising for detecting low frequency mutations, as true mutations could be distinguished from PCR errors or sequencing errors based on consensus among reads sharing same index. In an effort to develop a robust assay to detect from urine low-abundant bladder cancer cells carrying well-documented mutations, we have tested the idea first on a set of mock templates, with wild type and known mutants mixed at defined ratios. We have measured the combined error rate for PCR and Illumina sequencing at each nucleotide position of three exons, and demonstrated the power of a UID in distinguishing and correcting errors. In addition, we have demonstrated that PCR sampling bias, rather than PCR errors, challenges the UID-deep sequencing method in faithfully detecting low frequency mutation.
authors.affiliation: Department of Development, GENEWIZ LLC, 115 Corporate Blvd., South Plainfield, NJ, 07080, United States of America.
authors.forename: Ruqin
authors.initials: R
authors.lastname: Kou
authors.affiliation: Department of Development, GENEWIZ LLC, 115 Corporate Blvd., South Plainfield, NJ, 07080, United States of America.
authors.forename: Ham
authors.initials: H
authors.lastname: Lam
authors.affiliation: Department of Bioinformatics, GENEWIZ CN, 218 Xinghu Street, Suzhou, Jiangsu, 215123, China.
authors.forename: Hairong
authors.initials: H
authors.lastname: Duan
authors.affiliation: Department of Bioinformatics, GENEWIZ CN, 218 Xinghu Street, Suzhou, Jiangsu, 215123, China.
authors.forename: Li
authors.initials: L
authors.lastname: Ye
authors.affiliation: Department of Development, GENEWIZ LLC, 115 Corporate Blvd., South Plainfield, NJ, 07080, United States of America.
authors.forename: Narisra
authors.initials: N
authors.lastname: Jongkam
authors.affiliation: Department of Bioinformatics, GENEWIZ CN, 218 Xinghu Street, Suzhou, Jiangsu, 215123, China.
authors.forename: Weizhi
authors.initials: W
authors.lastname: Chen
authors.affiliation: Department of Development, GENEWIZ LLC, 115 Corporate Blvd., South Plainfield, NJ, 07080, United States of America.
authors.forename: Shifang
authors.initials: S
authors.lastname: Zhang
authors.affiliation: Department of Development, GENEWIZ LLC, 115 Corporate Blvd., South Plainfield, NJ, 07080, United States of America.
authors.forename: Shihong
authors.initials: S
authors.lastname: Li
mesh_terms: D001483:Base Sequence; D002894:Chromosomes, Human, Pair 4 / Q000235:genetics; D016000:Cluster Analysis; D004252:DNA Mutational Analysis; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D005091:Exons / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D059645:Mutation Rate; D009711:Nucleotides / Q000235:genetics; D016133:Polymerase Chain Reaction; D051498:Receptor, Fibroblast Growth Factor, Type 3 / Q000235:genetics; D015203:Reproducibility of Results; D017384:Sequence Deletion; D013698:Templates, Genetic
chemical_list: D009711:Nucleotides; C496365:FGFR3 protein, human; D051498:Receptor, Fibroblast Growth Factor, Type 3; D004259:DNA-Directed DNA Polymerase