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 |
Appears in Collections: | Elements Staff Publications |
Show full item record
Files in This Item:
File | Description | Size | Format | Access Settings | Version | |
---|---|---|---|---|---|---|
10_1038_srep33318.pdf | 2.69 MB | Adobe PDF | OPEN | None | View/Download |
This item is licensed under a Creative Commons License