Brown Julianne R, Roy Sunando, Ruis Christopher, Yara Romero Erika, Shah Divya, Williams Rachel, Breuer Judy
Microbiology, Virology, and Infection Control, Great Ormond Street Hospital for Children NHS Foundation Trust, London, United Kingdom NIHR Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust and University College London, London, United Kingdom
Division of Infection and Immunity, University College London, London, United Kingdom.
J Clin Microbiol. 2016 Oct;54(10):2530-7. doi: 10.1128/JCM.01052-16. Epub 2016 Aug 3.
Norovirus full-genome sequencing is challenging due to sequence heterogeneity among genomes. Previous methods have relied on PCR amplification, which is problematic due to primer design, and transcriptome sequencing (RNA-Seq), which nonspecifically sequences all RNA, including host and bacterial RNA, in stool specimens. Target enrichment uses a panel of custom-designed 120-mer RNA baits that are complementary to all publicly available norovirus sequences, with multiple baits targeting each position of the genome, which overcomes the challenge of primer design. Norovirus genomes are enriched from stool RNA extracts to minimize the sequencing of nontarget RNA. SureSelect target enrichment and Illumina sequencing were used to sequence full genomes from 507 norovirus-positive stool samples with reverse transcription-real-time PCR cycle threshold (CT) values of 10 to 43. Sequencing on an Illumina MiSeq system in batches of 48 generated, on average, 81% on-target reads per sample and 100% genome coverage with >12,000-fold read depth. Samples included genotypes GI.1, GI.2, GI.3, GI.6, GI.7, GII.1, GII.2, GII.3, GII.4, GII.5, GII.6, GII.7, GII.13, GII.14, and GII.17. When outliers were accounted for, we generated >80% genome coverage for all positive samples, regardless of CT values. A total of 164 samples were tested in parallel with conventional PCR genotyping of the capsid shell domain; 164/164 samples were successfully sequenced, compared to 158/164 samples that were amplified by PCR. Four of the samples that failed capsid PCR analysis had low titers, which suggests that target enrichment is more sensitive than gel-based PCR. Two samples failed PCR due to primer mismatches; target enrichment uses multiple baits targeting each position, thus accommodating sequence heterogeneity among norovirus genomes.
由于基因组之间的序列异质性，诺如病毒全基因组测序具有挑战性。以前的方法依赖于PCR扩增，由于引物设计存在问题，以及转录组测序（RNA-Seq），后者会对粪便样本中的所有RNA（包括宿主和细菌RNA）进行非特异性测序。靶向富集使用一组定制设计的120聚体RNA诱饵，这些诱饵与所有公开可用的诺如病毒序列互补，多个诱饵针对基因组的每个位置，从而克服了引物设计的挑战。从粪便RNA提取物中富集诺如病毒基因组，以尽量减少非靶标RNA的测序。使用SureSelect靶向富集和Illumina测序对507份诺如病毒阳性粪便样本的全基因组进行测序，这些样本的逆转录实时PCR循环阈值（CT）值为10至43。在Illumina MiSeq系统上以48个样本为一批进行测序，每个样本平均产生81%的靶向 reads，基因组覆盖率为100%，读深度>12000倍。样本包括GI.1、GI.2、GI.3、GI.6、GI.7、GII.1、GII.2、GII.3、GII.4、GII.5、GII.6、GII.7、GII.13、GII.14和GII.17基因型。当考虑异常值时，无论CT值如何，我们对所有阳性样本都产生了>80%的基因组覆盖率。总共164个样本与衣壳壳域的传统PCR基因分型进行了平行测试；164/164个样本成功测序，相比之下，158/164个样本通过PCR扩增。衣壳PCR分析失败的4个样本滴度较低，这表明靶向富集比基于凝胶的PCR更敏感。2个样本由于引物错配而PCR失败；靶向富集使用针对每个位置的多个诱饵，因此能够适应诺如病毒基因组之间的序列异质性。
