title: AmpliVar: mutation detection in high-throughput sequence from amplicon-based libraries.
abstract: Conventional means of identifying variants in high-throughput sequencing align each read against a reference sequence, and then call variants at each position. Here, we demonstrate an orthogonal means of identifying sequence variation by grouping the reads as amplicons prior to any alignment. We used AmpliVar to make key-value hashes of sequence reads and group reads as individual amplicons using a table of flanking sequences. Low-abundance reads were removed according to a selectable threshold, and reads above this threshold were aligned as groups, rather than as individual reads, permitting the use of sensitive alignment tools. We show that this approach is more sensitive, more specific, and more computationally efficient than comparable methods for the analysis of amplicon-based high-throughput sequencing data. The method can be extended to enable alignment-free confirmation of variants seen in hybridization capture target-enrichment data.
authors: Department of Pathology, The University of Melbourne, Parkville, Victoria, Australia.
authors: Arthur L
authors: AL
authors: Hsu
authors: Olga
authors: O
authors: Kondrashova
authors: Sebastian
authors: S
authors: Lunke
authors: Clare J
authors: CJ
authors: Love
authors: Cliff
authors: C
authors: Meldrum
authors: Renate
authors: R
authors: Marquis-Nicholson
authors: Greg
authors: G
authors: Corboy
authors: Kym
authors: K
authors: Pham
authors: Matthew
authors: M
authors: Wakefield
authors: Paul M
authors: PM
authors: Waring
authors: Graham R
authors: GR
authors: Taylor
mesh_terms: D019295:Computational Biology / Q000379:methods; D004252:DNA Mutational Analysis / Q000379:methods*; D015723:Gene Library; D014644:Genetic Variation; D023281:Genomics / Q000379:methods*; D060005:Genotyping Techniques; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D020407:Internet; D009154:Mutation; D021141:Nucleic Acid Amplification Techniques; D012984:Software
keywords: amplicon sequencing; grouped reads; mutation detection; next generation sequencing