The impact of sequencing depth on the inferred taxonomic composition and AMR gene content of metagenomic samples
The impact of sequencing depth on the inferred taxonomic composition and AMR gene content of metagenomic samples
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测序深度对宏基因组样本推断分类组成和AMR基因含量的影响
DOI：
10.1101/593301
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发表时间：
2019
期刊：
影响因子：
--
通讯作者：
Gweon H
中科院分区：
文献类型：
--
作者：
Gweon H
背景Shotgun宏基因组学越来越多地用于微生物群落的研究，特别是在不同动物和环境背景下的抗菌素耐药性（AMR）的研究。从鸟枪法宏基因组数据推断复杂群落样品的分类组成和AMR基因含量有许多不同的方法，但建立这些样品的最佳测序深度、数据处理和分析方法的工作很少。在这项研究中，我们使用鸟枪宏基因组学和测序的培养分离物从相同的样品来解决这些问题。我们对三个潜在的环境AMR基因库（猪盲肠、河流沉积物、污水）进行了采样，并在高深度（每个样本约2亿次读取）使用鸟枪宏基因组学对样本进行了测序。除此之外，我们从相同的样本中培养了肠杆菌科的单菌落分离株，并使用杂交测序（短读段和长读段）来创建高质量的组装体，以与宏基因组数据进行比较。为了自动化数据处理，我们开发了一个开源的软件管道，'ResPipe'.ResultsTaxonomic配置是更稳定的测序深度比AMR基因内容。每个样品100万个读段足以实现与完整分类组成< 1%的差异。然而，每个样品需要至少8000万个读段来恢复样品中存在的不同AMR基因家族的全部丰富度，并且在每个样品2亿个读段的流出物中仍发现AMR基因的额外等位基因多样性。使用基因长度和嗜热栖热菌DNA的外源性尖峰对AMR基因的读段数进行归一化，基本上改变了估计的基因丰度分布。虽然大多数的基因组内容从培养的菌株从流出物是可回收的使用鸟枪宏基因组学，这是不是猪盲肠或河流sediment.ConclusionsSequencing深度和剖析方法的情况下，可以严重影响剖析的多微生物动物和环境样品与鸟枪宏基因组学。培养分离株的测序和鸟枪宏基因组学都可以恢复使用其他方法无法识别的大量多样性。当通过将宏基因组读数映射到数据库来推断AMR基因含量或存在时，需要特别考虑。我们开发的开源软件管道ResPipe是免费提供的（ https://gitlab.com/hsgweon/ResPipe ).
BackgroundShotgun metagenomics is increasingly used to characterise microbial communities, particularly for the investigation of antimicrobial resistance (AMR) in different animal and environmental contexts. There are many different approaches for inferring the taxonomic composition and AMR gene content of complex community samples from shotgun metagenomic data, but there has been little work establishing the optimum sequencing depth, data processing and analysis methods for these samples. In this study we used shotgun metagenomics and sequencing of cultured isolates from the same samples to address these issues. We sampled three potential environmental AMR gene reservoirs (pig caeca, river sediment, effluent) and sequenced samples with shotgun metagenomics at high depth (~ 200 million reads per sample). Alongside this, we cultured single-colony isolates ofEnterobacteriaceaefrom the same samples and used hybrid sequencing (short- and long-reads) to create high-quality assemblies for comparison to the metagenomic data. To automate data processing, we developed an open-source software pipeline, ‘ResPipe’.ResultsTaxonomic profiling was much more stable to sequencing depth than AMR gene content. 1 million reads per sample was sufficient to achieve < 1% dissimilarity to the full taxonomic composition. However, at least 80 million reads per sample were required to recover the full richness of different AMR gene families present in the sample, and additional allelic diversity of AMR genes was still being discovered in effluent at 200 million reads per sample. Normalising the number of reads mapping to AMR genes using gene length and an exogenous spike ofThermus thermophilusDNA substantially changed the estimated gene abundance distributions. While the majority of genomic content from cultured isolates from effluent was recoverable using shotgun metagenomics, this was not the case for pig caeca or river sediment.ConclusionsSequencing depth and profiling method can critically affect the profiling of polymicrobial animal and environmental samples with shotgun metagenomics. Both sequencing of cultured isolates and shotgun metagenomics can recover substantial diversity that is not identified using the other methods. Particular consideration is required when inferring AMR gene content or presence by mapping metagenomic reads to a database. ResPipe, the open-source software pipeline we have developed, is freely available ( https://gitlab.com/hsgweon/ResPipe ).
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开放式细菌种群基因组学：BIGSDB软件，pubmlst.org网站及其应用。
影响因子：
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作者：
Jolley KA;Bray JE;Maiden MCJ
通讯作者：
Maiden MCJ
影响因子：
5.2
作者：
Vanessa Saliba
通讯作者：
Vanessa Saliba
影响因子：
4.4
作者：
DeSantis, T. Z.;Hugenholtz, P.;Andersen, G. L.
通讯作者：
Andersen, G. L.
影响因子：
4.6
作者：
Nordahl Petersen T;Rasmussen S;Hasman H;Carøe C;Bælum J;Schultz AC;Bergmark L;Svendsen CA;Lund O;Sicheritz-Pontén T;Aarestrup FM
通讯作者：
Aarestrup FM
影响因子：
48
作者：
Sczyrba A;Hofmann P;Belmann P;Koslicki D;Janssen S;Dröge J;Gregor I;Majda S;Fiedler J;Dahms E;Bremges A;Fritz A;Garrido-Oter R;Jørgensen TS;Shapiro N;Blood PD;Gurevich A;Bai Y;Turaev D;DeMaere MZ;Chikhi R;Nagarajan N;Quince C;Meyer F;Balvočiūtė M;Hansen LH;Sørensen SJ;Chia BKH;Denis B;Froula JL;Wang Z;Egan R;Don Kang D;Cook JJ;Deltel C;Beckstette M;Lemaitre C;Peterlongo P;Rizk G;Lavenier D;Wu YW;Singer SW;Jain C;Strous M;Klingenberg H;Meinicke P;Barton MD;Lingner T;Lin HH;Liao YC;Silva GGZ;Cuevas DA;Edwards RA;Saha S;Piro VC;Renard BY;Pop M;Klenk HP;Göker M;Kyrpides NC;Woyke T;Vorholt JA;Schulze-Lefert P;Rubin EM;Darling AE;Rattei T;McHardy AC
通讯作者：
McHardy AC