Contamination-controlled high-throughput whole genome sequencing for influenza A viruses using the MiSeq sequencer.
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.
Department of Laboratory Medicine, National University Hospital, National University Health System, Singapore.
Hong Kai
HK
Lee
Department of Laboratory Medicine, National University Hospital, National University Health System, Singapore.
Chun Kiat
CK
Lee
Department of Infection, Immunity, Inflammation, University of Leicester, Leicester, UK.|Clinical Microbiology, Leicester Royal Infirmary, Leicester, UK.
Julian Wei-Tze
JW
Tang
Department of Laboratory Medicine, National University Hospital, National University Health System, Singapore.
Tze Ping
TP
Loh
Department of Laboratory Medicine, National University Hospital, National University Health System, Singapore.|Department of Pathology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
Evelyn Siew-Chuan
ES
Koay
D001483:Base Sequence; D016679:Genome, Viral / Q000235:genetics*; D023281:Genomics; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D053122:Influenza A Virus, H3N2 Subtype / Q000235:genetics* / Q000472:pathogenicity; D007251:Influenza, Human / Q000235:genetics* / Q000821:virology; D010802:Phylogeny; D017422:Sequence Analysis, DNA; D000073336:Whole Genome Sequencing / Q000379:methods*