Culture-independent detection and characterisation of Mycobacterium tuberculosis and M. africanum in sputum samples using shotgun metagenomics on a benchtop sequencer.
Culture-independent detection and characterisation of Mycobacterium tuberculosis and M. africanum in sputum samples using shotgun metagenomics on a benchtop sequencer.
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作者：
Doughty EL;Sergeant MJ;Adetifa I;Antonio M;Pallen MJ
结核病仍然是一个主要的全球健康问题。实验室诊断方法，使有效的，早期发现的情况下，是中央结核病管理的个人病人和社区。自19世纪80年代以来，结核病的实验室诊断主要依靠显微镜和培养。然而，显微镜检查无法提供物种或谱系水平的鉴定，并且用于诊断结核病的基于培养的工作流程仍然复杂、昂贵、缓慢、技术要求高并且难以处理混合感染。因此，我们探索了鸟枪宏基因组学的潜力，即在没有培养或靶特异性扩增或捕获的情况下对样本进行DNA测序，以检测和鉴定来自西非冈比亚的痰涂片阳性痰样本中的结核分枝杆菌复合体菌株。使用差异裂解方案，然后使用基于试剂盒的DNA提取方法，在台式测序仪Illumina MiSeq上进行测序，研究了8份涂片和培养阳性痰液样本。每个大肠杆菌来源的宏基因组中的序列读数的数量范围为989，442至2，818，238。针对人类基因组的每个宏基因组映射中的读段比例范围为20%至99%。我们检测到了M.在所有8个样品中，H37 Rv参考基因组的覆盖范围为0.002X至0.7X。通过分析大序列多态性（插入元件IS 6110的缺失和位置）和单核苷酸多态性（SNP）的分布，我们能够将8个宏基因组衍生的基因组中的7个分配给M.肺结核综合征两个宏基因组衍生的分枝杆菌基因组被分配给M。africanum，一个主要局限于西非的种;其他可以被分配属于“现代”M分支内的谱系T，H或LAM。结核菌株我们已经提供了鸟枪宏基因组学可用于检测和分析M的原理证明。在没有培养或靶特异性扩增或捕获的情况下，使用可访问的台式测序平台Illumina MiSeq和相对简单的DNA提取、测序和生物信息学方案，从痰液样品中提取结核病序列。在我们手中，痰液宏基因组学还没有提供足够的覆盖深度，以允许基于序列的敏感性测试;还有待确定是否单独改进DNA提取方案可以提供这一点，或者是否需要培养，捕获或扩增步骤。尽管如此，我们可以预见一个临界点，即统一的自动化宏基因组学工作流程可能开始与诊断微生物学实验室目前使用的大量方法竞争。
Tuberculosis remains a major global health problem. Laboratory diagnostic methods that allow effective, early detection of cases are central to management of tuberculosis in the individual patient and in the community. Since the 1880s, laboratory diagnosis of tuberculosis has relied primarily on microscopy and culture. However, microscopy fails to provide species- or lineage-level identification and culture-based workflows for diagnosis of tuberculosis remain complex, expensive, slow, technically demanding and poorly able to handle mixed infections. We therefore explored the potential of shotgun metagenomics, sequencing of DNA from samples without culture or target-specific amplification or capture, to detect and characterise strains from the Mycobacterium tuberculosis complex in smear-positive sputum samples obtained from The Gambia in West Africa. Eight smear- and culture-positive sputum samples were investigated using a differential-lysis protocol followed by a kit-based DNA extraction method, with sequencing performed on a benchtop sequencing instrument, the Illumina MiSeq. The number of sequence reads in each sputum-derived metagenome ranged from 989,442 to 2,818,238. The proportion of reads in each metagenome mapping against the human genome ranged from 20% to 99%. We were able to detect sequences from the M. tuberculosis complex in all eight samples, with coverage of the H37Rv reference genome ranging from 0.002X to 0.7X. By analysing the distribution of large sequence polymorphisms (deletions and the locations of the insertion element IS6110) and single nucleotide polymorphisms (SNPs), we were able to assign seven of eight metagenome-derived genomes to a species and lineage within the M. tuberculosis complex. Two metagenome-derived mycobacterial genomes were assigned to M. africanum, a species largely confined to West Africa; the others that could be assigned belonged to lineages T, H or LAM within the clade of “modern” M. tuberculosis strains. We have provided proof of principle that shotgun metagenomics can be used to detect and characterise M. tuberculosis sequences from sputum samples without culture or target-specific amplification or capture, using an accessible benchtop-sequencing platform, the Illumina MiSeq, and relatively simple DNA extraction, sequencing and bioinformatics protocols. In our hands, sputum metagenomics does not yet deliver sufficient depth of coverage to allow sequence-based sensitivity testing; it remains to be determined whether improvements in DNA extraction protocols alone can deliver this or whether culture, capture or amplification steps will be required. Nonetheless, we can foresee a tipping point when a unified automated metagenomics-based workflow might start to compete with the plethora of methods currently in use in the diagnostic microbiology laboratory.
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DOI：
10.1016/j.tube.2014.02.005
发表时间：
2014-05
期刊：
Tuberculosis (Edinburgh, Scotland)
影响因子：
--
作者：
Coll F;Preston M;Guerra-Assunção JA;Hill-Cawthorn G;Harris D;Perdigão J;Viveiros M;Portugal I;Drobniewski F;Gagneux S;Glynn JR;Pain A;Parkhill J;McNerney R;Martin N;Clark TG
通讯作者：
Clark TG
影响因子：
--
作者：
de Jong, Bouke C.;Adetifa, Ifedayo;Adegbola, Richard A.
通讯作者：
Adegbola, Richard A.
影响因子：
9.4
作者：
Kamerbeek, J;Schouls, L;vanEmbden, J
通讯作者：
vanEmbden, J
影响因子：
--
作者：
Jagielski T;van Ingen J;Rastogi N;Dziadek J;Mazur PK;Bielecki J
通讯作者：
Bielecki J
DOI：
10.5588/ijtld.12.0180
发表时间：
2012-07-01
影响因子：
4
作者：
Drobniewski, F.;Nikolayevskyy, V.;Papaventsis, D.
通讯作者：
Papaventsis, D.