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

香港中文大學研究人員

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摘要Background 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.
著者KWOK Tsoi-tung, CHAN Chi-wai
會議名稱INFECTION 2017
會議開始日20.09.2017
會議完結日21.09.2017
會議地點Hong Kong
會議國家/地區香港
出版年份2017
語言美式英語

上次更新時間 2018-10-05 於 15:03