Contamination-controlled high-throughput whole genome sequencing for influenza A viruses using the MiSeq sequencer

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.

Divergent evolutionary trajectories of influenza B viruses underlie their contemporaneous epidemic activity

“…For Sanger sequencing, the viruses were passaged in Madin-Darby Canine Kidney SIAT-1 cells and sequenced using previously published primers (31). NGS sequencing was performed on libraries of cDNA derived from viral RNA extracted directly from the swabs and followed by RT-PCR amplification of whole genome of influenza B (32,33). The PCR products were quantified using the Qubit dsDNA HS Assay kits (Thermo Fisher Scientific) and subsequently diluted to 1 ng/μL for library preparation using the Nextera XT DNA library preparation kits (Illumina).…”

Improved detection of influenza A virus from blue‐winged teals by sequencing directly from swab material

“…Previously, DIPs were detected in influenza viruses obtained from naturally infected human and chicken samples (Chambers & Webster, 1987;Saira et al, 2013). Within NGS data, DIPs are usually inferred by the formation a valley-like shape in the coverage distribution plot, produced by a bias of reads toward the 3′ and 5′ ends of the gene segments (Lee, Lee, Tang, Loh, & Koay, 2016;Saira et al, 2013). We compared the coverage distribution of PB2, PB1, and PA produced by swab-NGS and VI-NGS.…”

Molecular insights into evolution, mutations and receptor-binding specificity of influenza A and B viruses from outpatients and hospitalized patients in Singapore

“…Illumina MiSeq Next Generation Sequencer was employed to sequence the full genomes of influenza A, while FluSeq v1.0 was used for genome assembly (Lee et al, 2016). Sanger sequencing was employed to validate the HA1 segments of influenza A, and to sequence the genomes of influenza B. A/H3N2 primers (Lee et al, 2013), A/H1N1 primers (Deng et al, 2015) and 18 sets of B primers (Tewawong et al, 2015a) were used for sequencing.…”