A modified RNA-Seq approach for whole genome sequencing of RNA viruses from faecal and blood samples.
A modified RNA-Seq approach for whole genome sequencing of RNA viruses from faecal and blood samples.
复制标题
DOI：
10.1371/journal.pone.0066129
复制
发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Piazza P
中科院分区：
文献类型：
--
作者：
Batty EM;Wong TH;Trebes A;Argoud K;Attar M;Buck D;Ip CL;Golubchik T;Cule M;Bowden R;Manganis C;Klenerman P;Barnes E;Walker AS;Wyllie DH;Wilson DJ;Dingle KE;Peto TE;Crook DW;Piazza P
迄今为止，许多具有临床意义的RNA病毒的大规模测序由于其高群体分子变异而变得复杂，这给聚合酶链反应和测序引物设计带来了挑战。许多RNA病毒也很难或目前不可能培养，这严重限制了可用起始材料的数量和纯度。在这里，我们描述了一种基于RNA鸟枪测序（也称为RNA- seq）的简单、新颖、高通量的诺如病毒和丙型肝炎病毒全基因组序列测定方法。我们在Illumina MiSeq台式测序仪上对来自粪便的三个诺如病毒样本和两个来自血液的丙型肝炎病毒样本进行了测序，证明了这种方法的有效性。超过97%的参考基因组被恢复。与Sanger测序相比，我们的方法对诺如病毒（从Sanger测序获得的2个诺如病毒基因组中）的14,019个核苷酸（nt）没有核苷酸差异，对丙型肝炎病毒（9,542 nt）的8个变异（每1,193 nt 1个变异）。三种诺如病毒样本分别有2个、3个和2个不同的位置称为杂合，而两种丙型肝炎病毒样本分别有117个和131个位置称为杂合。为了证实我们的样品和文库制备可以达到真正的高通量，我们在Illumina HiSeq 2000测序仪上制备并测序了另外77个诺如病毒样品，除一个样品外，其余样品中参考基因组的回收率为50%。在16个样本中，Sanger和我们定制的RNA-Seq方法在118,757 nt中没有观察到差异。通过生成不受引物特异性扩增或富集影响的病毒基因组序列，该方法为大规模、可负担得起的RNA病毒研究提供了前景，这种研究可以在不久的将来适应常规诊断实验室工作流程，具有直接表征宿主内病毒多样性的潜力。
To date, very large scale sequencing of many clinically important RNA viruses has been complicated by their high population molecular variation, which creates challenges for polymerase chain reaction and sequencing primer design. Many RNA viruses are also difficult or currently not possible to culture, severely limiting the amount and purity of available starting material. Here, we describe a simple, novel, high-throughput approach to Norovirus and Hepatitis C virus whole genome sequence determination based on RNA shotgun sequencing (also known as RNA-Seq). We demonstrate the effectiveness of this method by sequencing three Norovirus samples from faeces and two Hepatitis C virus samples from blood, on an Illumina MiSeq benchtop sequencer. More than 97% of reference genomes were recovered. Compared with Sanger sequencing, our method had no nucleotide differences in 14,019 nucleotides (nt) for Noroviruses (from a total of 2 Norovirus genomes obtained with Sanger sequencing), and 8 variants in 9,542 nt for Hepatitis C virus (1 variant per 1,193 nt). The three Norovirus samples had 2, 3, and 2 distinct positions called as heterozygous, while the two Hepatitis C virus samples had 117 and 131 positions called as heterozygous. To confirm that our sample and library preparation could be scaled to true high-throughput, we prepared and sequenced an additional 77 Norovirus samples in a single batch on an Illumina HiSeq 2000 sequencer, recovering >90% of the reference genome in all but one sample. No discrepancies were observed across 118,757 nt compared between Sanger and our custom RNA-Seq method in 16 samples. By generating viral genomic sequences that are not biased by primer-specific amplification or enrichment, this method offers the prospect of large-scale, affordable studies of RNA viruses which could be adapted to routine diagnostic laboratory workflows in the near future, with the potential to directly characterize within-host viral diversity.
登录
查看更多内容
影响因子：
3
作者：
Prosperi MC;Prosperi L;Bruselles A;Abbate I;Rozera G;Vincenti D;Solmone MC;Capobianchi MR;Ulivi G
通讯作者：
Ulivi G
影响因子：
14.9
作者：
Hansen KD;Brenner SE;Dudoit S
通讯作者：
Dudoit S
影响因子：
9.4
作者：
Croville, Guillaume;Soubies, Sebastien Mathieu;Guerin, Jean-Luc
通讯作者：
Guerin, Jean-Luc
影响因子：
7
作者：
Lunter, Gerton;Goodson, Martin
通讯作者：
Goodson, Martin
影响因子：
11.8
作者：
Patel, Manish M.;Widdowson, Marc-Alain;Glass, Roger I.;Akazawa, Kenichiro;Vinje, Jan;Parashar, Umesh D.
通讯作者：
Parashar, Umesh D.