Please use this identifier to cite or link to this item: https://doi.org/10.1038/srep33318
Title: Contamination-controlled high-throughput whole genome sequencing for influenza A viruses using the MiSeq sequencer
Authors: Lee, H.K
Lee, C.K
Tang, J.W.-T
Loh, T.P 
Koay, E.S.-C 
Keywords: DNA sequence
genetics
genomics
high throughput sequencing
human
influenza
Influenza A virus (H3N2)
nucleotide sequence
pathogenicity
phylogeny
procedures
virology
virus genome
whole genome sequencing
Base Sequence
Genome, Viral
Genomics
High-Throughput Nucleotide Sequencing
Humans
Influenza A Virus, H3N2 Subtype
Influenza, Human
Phylogeny
Sequence Analysis, DNA
Whole Genome Sequencing
Issue Date: 2016
Citation: Lee, H.K, Lee, C.K, Tang, J.W.-T, Loh, T.P, Koay, E.S.-C (2016). Contamination-controlled high-throughput whole genome sequencing for influenza A viruses using the MiSeq sequencer. Scientific Reports 6 : 33318. ScholarBank@NUS Repository. https://doi.org/10.1038/srep33318
Rights: Attribution 4.0 International
Abstract: Accurate full-length genomic sequences are important for viral phylogenetic studies. We developed a targeted high-throughput whole genome sequencing (HT-WGS) method for influenza A viruses, which utilized an enzymatic cleavage-based approach, the Nextera XT DNA library preparation kit, for library preparation. The entire library preparation workflow was adapted for the Sentosa SX101, a liquid handling platform, to automate this labor-intensive step. As the enzymatic cleavage-based approach generates low coverage reads at both ends of the cleaved products, we corrected this loss of sequencing coverage at the termini by introducing modified primers during the targeted amplification step to generate full-length influenza A sequences with even coverage across the whole genome. Another challenge of targeted HTS is the risk of specimen-to-specimen cross-contamination during the library preparation step that results in the calling of false-positive minority variants. We included an in-run, negative system control to capture contamination reads that may be generated during the liquid handling procedures. The upper limits of 99.99% prediction intervals of the contamination rate were adopted as cut-off values of contamination reads. Here, 148 influenza A/H3N2 samples were sequenced using the HTS protocol and were compared against a Sanger-based sequencing method. Our data showed that the rate of specimen-to-specimen cross-contamination was highly significant in HTS. © 2016 The Author(s).
Source Title: Scientific Reports
URI: https://scholarbank.nus.edu.sg/handle/10635/178864
ISSN: 20452322
DOI: 10.1038/srep33318
Rights: Attribution 4.0 International
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