Metagenomic next-generation viral genome sequencing on clinical samples
Refereed conference paper presented and published in conference proceedings


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AbstractBackground and Aims:
Advent of next-generation sequencing (NGS) technology has unprecedentedly improved costs, scale and speed of genome characterization. Whole genome sequencing of pathogens, one of the most revolutionary NGS applications, has become increasingly prominent for not only basic research but also clinical diagnosis of acute infections. Here, we aim to evaluate the performance of metagenomic viral genome sequencing on clinical samples.

Method:
Norovirus-positive 10% (wt/vol) stool suspension in phosphate-buffered saline were centrifuged and filtered, followed by pre-extraction nuclease treatment. Viral RNA protected in intact virions was extracted and purified using the QIAamp Viral RNA Mini Kit. Reverse transcription was performed using tagged random octamers and SuperScript III reverse transcriptase. Second-strand cDNA was synthesized by Klenow fragment, followed by random cDNA amplification. Sample libraries were prepared using the Nextera XT DNA library prep kit v2 and sequenced using the MiSeq platform to produce 2x75 bp paired-end reads. Quality trimming were performed using Trimmomatic v0.36. Reference mapping was performed using Geneious R10.

Results:
A total of 97 norovirus strains from hospitalized patients with acute gastroenteritis were sequenced. Sample viral load (diagnostic RT-qPCR cycle threshold value) ranged from 5.5-19.6 (median 14.4). Median percentage on-target read (%OTR) was 73%. Almost 90% of our samples had a percentage genome coverage (%GC) equal or higher than 90%. Both %GC (P<0.0001, Spearman r=-0.5489) and %OTR (P<0.0001, Spearman r=‑0.3904) were significantly associated with input viral load. Median mean sequencing depth of 7,344x indicated this method was suitable for intrahost analysis.

Conclusion:
Our metagenomic viral genome sequencing method gives a high sequencing quality in terms of high %OTR and %GC. Further integration of de novo assembly would make this method a powerful tool for genome characterization of novel viruses.
All Author(s) ListKWOK Tsoi-tung, CHAN Chi-wai
Name of ConferenceINFECTION 2017
Start Date of Conference20/09/2017
End Date of Conference21/09/2017
Place of ConferenceHong Kong
Country/Region of ConferenceHong Kong
Year2017
LanguagesEnglish-United States

Last updated on 2018-10-05 at 15:03