Optimisation of 16S rRNA gut microbiota profiling of extremely low birth weight infants.
Optimisation of 16S rRNA gut microbiota profiling of extremely low birth weight infants.
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DOI：
10.1186/s12864-017-4229-x
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发表时间：
2017-11-02
期刊：
影响因子：
4.4
通讯作者：
Hall LJ
中科院分区：
文献类型：
--
作者：
Alcon-Giner C;Caim S;Mitra S;Ketskemety J;Wegmann U;Wain J;Belteki G;Clarke P;Hall LJ
早产婴儿，特别是极低出生体重婴儿（ELBW）已经改变了肠道微生物群落。孕产妇健康、肠道不成熟、分娩方式和抗生素治疗等因素与微生物群紊乱有关，并与坏死性小肠结肠炎等某些疾病的风险增加有关。因此，需要通过方法标准化来最佳地表征该高危人群的微生物特征，特别是研究微生物群疗法（例如益生菌补充剂）对社区特征和健康结果的影响。使用 16S rRNA 基因对粪便样本进行分析是一种经济高效的方法，适用于大规模临床研究，以深入了解肠道微生物群，并且还可以对样本数量受到影响的群体（例如 ELBW 婴儿）进行表征。然而，DNA 提取方法和目标 16S rRNA 区域可以显着改变获得的细菌群落特征，因此使研究之间的比较变得混乱。因此，我们试图优化 16S rRNA 分析方案，以实现 ELBW 婴儿粪便样本研究的标准化，无论是否补充益生菌。使用ELBW粪便样本，我们比较了三种不同的DNA提取方法，随后PCR扩增并测序了16S rRNA基因的三个高变区（V1 + V2 + V3）、（V4 + V5）和（V6 + V7 + V8），并比较了两种生物信息学方法来分析结果（OTU和配对末端）。配对鸟枪法宏基因组学被用作“黄金标准”。结果表明，最佳细菌 DNA 提取需要更长的敲珠步骤，并且测序区域 (V1 + V2 + V3) 和 (V6 + V7 + V8) 提供了最具代表性的分类学特征，这通过鸟枪法分析得到了证实。使用 (V4 + V5) 区域测序的样本被发现在包括双歧杆菌在内的特定类群中代表性不足，并且多样性概况发生了改变。本研究中使用的两种生物信息学 16S rRNA 管道（OTU 和配对末端）在属水平上呈现出相似的分类学特征。我们确定，从 ELBW 粪便样本中提取 DNA，特别是那些接受益生菌补充剂的婴儿，应该包括长时间的敲打步骤。此外，使用 16S rRNA (V1 + V2 + V3) 和 (V6 + V7 + V8) 区域可以可靠地表示 ELBW 微生物群概况，而包含 (V4 + V5) 区域可能不适合双歧杆菌构成常驻微生物群成员的研究。本文的在线版本 (10.1186/s12864-017-4229-x) 包含补充材料，可供授权用户使用。
Infants born prematurely, particularly extremely low birth weight infants (ELBW) have altered gut microbial communities. Factors such as maternal health, gut immaturity, delivery mode, and antibiotic treatments are associated with microbiota disturbances, and are linked to an increased risk of certain diseases such as necrotising enterocolitis. Therefore, there is a requirement to optimally characterise microbial profiles in this at-risk cohort, via standardisation of methods, particularly for studying the influence of microbiota therapies (e.g. probiotic supplementation) on community profiles and health outcomes. Profiling of faecal samples using the 16S rRNA gene is a cost-efficient method for large-scale clinical studies to gain insights into the gut microbiota and additionally allows characterisation of cohorts were sample quantities are compromised (e.g. ELBW infants). However, DNA extraction method, and the 16S rRNA region targeted can significantly change bacterial community profiles obtained, and so confound comparisons between studies. Thus, we sought to optimise a 16S rRNA profiling protocol to allow standardisation for studying ELBW infant faecal samples, with or without probiotic supplementation. Using ELBW faecal samples, we compared three different DNA extraction methods, and subsequently PCR amplified and sequenced three hypervariable regions of the 16S rRNA gene (V1 + V2 + V3), (V4 + V5) and (V6 + V7 + V8), and compared two bioinformatics approaches to analyse results (OTU and paired end). Paired shotgun metagenomics was used as a ‘gold-standard’. Results indicated a longer bead-beating step was required for optimal bacterial DNA extraction and that sequencing regions (V1 + V2 + V3) and (V6 + V7 + V8) provided the most representative taxonomic profiles, which was confirmed via shotgun analysis. Samples sequenced using the (V4 + V5) region were found to be underrepresented in specific taxa including Bifidobacterium, and had altered diversity profiles. Both bioinformatics 16S rRNA pipelines used in this study (OTU and paired end) presented similar taxonomic profiles at genus level. We determined that DNA extraction from ELBW faecal samples, particularly those infants receiving probiotic supplementation, should include a prolonged beat-beating step. Furthermore, use of the 16S rRNA (V1 + V2 + V3) and (V6 + V7 + V8) regions provides reliable representation of ELBW microbiota profiles, while inclusion of the (V4 + V5) region may not be appropriate for studies where Bifidobacterium constitutes a resident microbiota member. The online version of this article (10.1186/s12864-017-4229-x) contains supplementary material, which is available to authorized users.
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