Ubiquitous microbial contaminants associated with scientific ocean drilling.

Access microbiology Pub Date : 2025-03-07 eCollection Date: 2025-01-01 DOI:10.1099/acmi.0.000865.v3
Jessica M Labonté, Kathryn L Campbell, Jordan R Walker, Milena A Rodriguez-Pilco
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引用次数: 0

Abstract

Deep-sea subsurface samples typically have low microbial biomass, making them more susceptible to contamination. Potential contaminants can be introduced during any step of the scientific process, including drilling (contamination from the drilling fluid and lubricants and seawater); sample preparation (contamination from air and human handling); and DNA extraction, amplification and sequencing (contamination from reagents). The International Ocean Discovery Program (IODP) samples that are dedicated to microbiological analyses (known as MBIO samples) are routinely tested for contamination by injecting known concentrations of fluorescent microspheres or tracers directly into the drilling fluids. These tracers are a great tool to determine the level of drilling fluid contamination on board, but they are not consistently used, and contamination of the samples could occur during other steps of sample processing and analysis. Moreover, there is an increased risk of contamination dominating the results of microbial surveys using PCR amplification of marker genes. Here, we built a database of common contaminants through the screening of contamination controls from available 16S rRNA gene amplicon datasets from past IODP expeditions. These controls included various lubricants used on board, drilling fluids, seawater, DNA extraction blanks and PCR blanks. The order Burkholderiales dominated most of the very low biomass samples, including negative controls, indicating the order's ubiquity and its potential to be overamplified with common 16S rRNA amplification protocols. We amplified the 16S rRNA gene from preserved IODP legacy microbiological core samples and tested their level of contamination using the database. We also looked at published studies that did not sequence negative controls. Our results demonstrate that the type of drilling, amount of manipulation of the sample prior to preservation and sample depth, often associated with biomass, can influence the level of contamination within subsurface samples. This work provides an analysis framework for microbial taxonomic survey studies from low biomass subsurface samples for future scientific ocean drilling expeditions.

与科学海洋钻探相关的无处不在的微生物污染物。
深海地下样本的微生物量通常较低,因此更容易受到污染。潜在的污染物可以在科学过程的任何步骤中引入,包括钻井(来自钻井液、润滑剂和海水的污染);样品制备(空气污染和人为处理);DNA提取、扩增和测序(试剂污染)。国际海洋发现计划(IODP)用于微生物分析的样品(称为MBIO样品)通常通过将已知浓度的荧光微球或示踪剂直接注入钻井液中进行污染检测。这些示踪剂是确定船上钻井液污染水平的好工具,但它们并没有一直使用,样品污染可能在样品处理和分析的其他步骤中发生。此外,使用PCR扩增标记基因的微生物调查结果中污染的风险增加。在这里,我们通过筛选来自过去IODP探险中可用的16S rRNA基因扩增子数据集的污染控制,建立了一个常见污染物的数据库。这些控制包括船上使用的各种润滑剂、钻井液、海水、DNA提取空白和PCR空白。包括阴性对照在内的大部分极低生物量样本中,Burkholderiales占主导地位,这表明该目的普遍存在及其可能被常见的16S rRNA扩增方案过度扩增。我们从保存的IODP遗留微生物核心样本中扩增了16S rRNA基因,并使用数据库测试了它们的污染水平。我们还查看了未对阴性对照进行排序的已发表研究。我们的研究结果表明,钻孔类型、保存前样品的操作量和样品深度(通常与生物量有关)可以影响地下样品中的污染水平。本研究为今后的海洋钻探科学考察提供了低生物量地下样品微生物分类调查研究的分析框架。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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