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首页> 外文期刊>Investigative Genetics >Accurate, rapid and high-throughput detection of strain-specific polymorphisms in Bacillus anthracis and Yersinia pestis by next-generation sequencing
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Accurate, rapid and high-throughput detection of strain-specific polymorphisms in Bacillus anthracis and Yersinia pestis by next-generation sequencing

机译：下一代测序技术可准确，快速，高通量检测炭疽芽孢杆菌和鼠疫耶尔森氏菌中的菌株特异性多态性

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Background In the event of biocrimes or infectious disease outbreaks, high-resolution genetic characterization for identifying the agent and attributing it to a specific source can be crucial for an effective response. Until recently, in-depth genetic characterization required expensive and time-consuming Sanger sequencing of a few strains, followed by genotyping of a small number of marker loci in a panel of isolates at or by gel-based approaches such as pulsed field gel electrophoresis, which by necessity ignores most of the genome. Next-generation, massively parallel sequencing (MPS) technology (specifically the Applied Biosystems sequencing by oligonucleotide ligation and detection (SOLiD?) system) is a powerful investigative tool for rapid, cost-effective and parallel microbial whole-genome characterization. Results To demonstrate the utility of MPS for whole-genome typing of monomorphic pathogens, four Bacillus anthracis and four Yersinia pestis strains were sequenced in parallel. Reads were aligned to complete reference genomes, and genomic variations were identified. Resequencing of the B. anthracis Ames ancestor strain detected no false-positive single-nucleotide polymorphisms (SNPs), and mapping of reads to the Sterne strain correctly identified 98% of the 133 SNPs that are not clustered or associated with repeats. Three geographically distinct B. anthracis strains from the A branch lineage were found to have between 352 and 471 SNPs each, relative to the Ames genome, and one strain harbored a genomic amplification. Sequencing of four Y. pestis strains from the Orientalis lineage identified between 20 and 54 SNPs per strain relative to the CO92 genome, with the single Bolivian isolate having approximately twice as many SNPs as the three more closely related North American strains. Coverage plotting also revealed a common deletion in two strains and an amplification in the Bolivian strain that appear to be due to insertion element-mediated recombination events. Most private SNPs (that is, a, variant found in only one strain in this set) selected for validation by Sanger sequencing were confirmed, although rare false-positive SNPs were associated with variable nucleotide tandem repeats. Conclusions The high-throughput, multiplexing capability, and accuracy of this system make it suitable for rapid whole-genome typing of microbial pathogens during a forensic or epidemiological investigation. By interrogating nearly every base of the genome, rare polymorphisms can be reliably discovered, thus facilitating high-resolution strain tracking and strengthening forensic attribution.
机译：背景技术在发生生物犯罪或传染病暴发的情况下，用于鉴定病原并将其归因于特定来源的高分辨率遗传特征对于有效应对至关重要。直到最近，深入的遗传表征都需要对几种菌株进行昂贵且耗时的Sanger测序，然后通过或基于凝胶的方法（例如脉冲场凝胶电泳）对一组分离物中的少量标记位点进行基因分型，它必然会忽略大部分基因组。下一代大规模并行测序（MPS）技术（特别是Applied Biosystems通过寡核苷酸连接和检测测序（SOLiD？）系统）是一种功能强大的研究工具，可用于快速，经济高效且并行的微生物全基因组表征。结果为了证明MPS在单基因病原体全基因组分型中的实用性，对4株炭疽芽孢杆菌和4株鼠疫耶尔森菌进行了平行测序。将阅读物比对以完成完整的参考基因组，并鉴定基因组变异。炭疽杆菌Ames祖先菌株的重测序未检测到假阳性单核苷酸多态性（SNP），并且将读图映射到Sterne菌株正确鉴定了133个SNP中没有聚类或与重复相关的98％。发现相对于Ames基因组，来自A分支谱系的三个地理上不同的炭疽芽孢杆菌菌株各自具有352至471个SNP，并且一个菌株具有基因组扩增。相对于CO92基因组，来自Orientalis谱系的四种鼠疫耶尔森氏菌菌株的测序确定每个菌株介于20到54个SNP之间，单个玻利维亚分离株的SNP大约是三个更紧密相关的北美菌株的两倍。覆盖图还显示了两个菌株中的常见缺失和玻利维亚菌株中的扩增，这似乎是由于插入元件介导的重组事件所致。尽管通过罕见的假阳性单核苷酸多态性与可变核苷酸串联重复序列相关联，但仍确认了通过Sanger测序验证选择的大多数私有SNP（即在该组中仅一个菌株中发现的变体）。结论该系统的高通量，多路复用能力和准确性使其适用于法医或流行病学调查期间微生物病原体的快速全基因组分型。通过询问基因组的几乎每个碱基，可以可靠地发现罕见的多态性，从而有助于高分辨率菌株追踪和加强法医鉴定。

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