VSITA，一种改进的病毒病原体鉴定中的靶标扩增方法。

VSITA, an Improved Approach of Target Amplification in the Identification of Viral Pathogens.

机构信息

Key Laboratory for Medical Virology, National Health and Family Planning Commission, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China.

State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100730, China.

出版信息

Biomed Environ Sci. 2018 Apr;31(4):272-279. doi: 10.3967/bes2018.035.

DOI:10.3967/bes2018.035

PMID:29773090

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7135048/

Abstract

OBJECTIVE

Unbiased next generation sequencing (NGS) is susceptible to interference from host or environmental sequences. Consequently, background depletion and virome enrichment techniques are usually needed for clinical samples where viral load is much lower than background sequences.

METHODS

A viral Sequence Independent Targeted Amplification (VSITA) approach using a set of non-ribosomal and virus-enriched octamers (V8) was developed and compared with traditionally used random hexamers (N6). Forty-five archived clinical samples of different types were used in parallel to compare the V8 and N6 enrichment performance of viral sequences and removal performance of ribosomal sequences in the step of reverse transcription followed by quantitative PCR (qPCR). Ten sera samples from patients with fever of unknown origin and 10 feces samples from patients with diarrhea of unknown origin were used in comparison of V8 and N6 enrichment performance following NGS analysis.

RESULTS

A minimum 30 hexamers matching to viral reference sequences (sense and antisense) were selected from a dataset of random 4,096 (46) hexamers (N6). Two random nucleotides were added to the 5' end of the selected hexamers, and 480 (30 × 42) octamers (V8) were obtained. In general, VSITA approach showed higher enrichment of virus-targeted cDNA and enhanced ability to remove unwanted ribosomal sequences in the majorities of 45 predefined clinical samples. Moreover, VSITA combined with NGS enabled to detect not only more viruses but also achieve more viral reads hit and higher viral genome coverage in 20 clinical samples with diarrhea or fever of unknown origin.

CONCLUSION

The VSITA approach designed in this study is demonstrated to possess higher sensitivity and broader genome coverage than traditionally used random hexamers in the NGS-based identification of viral pathogens directly from clinical samples.

摘要

目的

无偏下一代测序（NGS）易受宿主或环境序列的干扰。因此，对于病毒载量远低于背景序列的临床样本，通常需要进行背景耗竭和病毒富集技术。

方法

开发了一种使用非核糖体和富含病毒的八聚体（V8）的病毒序列独立靶向扩增（VSITA）方法，并与传统使用的随机六聚体（N6）进行了比较。同时使用 45 个不同类型的存档临床样本，比较 V8 和 N6 对病毒序列的富集性能，以及在逆转录后定量 PCR（qPCR）步骤中去除核糖体序列的性能。将 10 份发热原因不明的血清样本和 10 份腹泻原因不明的粪便样本用于比较 NGS 分析后 V8 和 N6 的富集性能。

结果

从随机 4096 个（46）六聚体（N6）数据集中选择了至少 30 个与病毒参考序列（有义和反义）匹配的六聚体。在选定的六聚体 5'端添加了两个随机核苷酸，得到 480 个（30×42）八聚体（V8）。一般来说，VSITA 方法在大多数 45 个预定义的临床样本中，显示出更高的病毒靶向 cDNA 富集能力，并且能够更好地去除不需要的核糖体序列。此外，VSITA 与 NGS 相结合，不仅能够在腹泻或发热原因不明的 20 个临床样本中检测到更多的病毒，而且还能够实现更多的病毒读段命中和更高的病毒基因组覆盖率。