Table_1_Repetitive Sampling and Control Threshold Improve 16S rRNA Gene Sequencing Results From Produced Waters Associated With Hydraulically Fractured Shale.xlsx

Sequencing microbial DNA from deep subsurface environments is complicated by a number of issues ranging from contamination to non-reproducible results. Many samples obtained from these environments – which are of great interest due to the potential to stimulate microbial methane generation – contain low biomass. Therefore, samples from these environments are difficult to study as sequencing results can be easily impacted by contamination. In this case, the low amount of sample biomass may be effectively swamped by the contaminating DNA and generate misleading results. Additionally, performing field work in these environments can be difficult, as researchers generally have limited access to and time on site. Therefore, optimizing a sampling plan to produce the best results while collecting the greatest number of samples over a short period of time is ideal. This study aimed to recommend an adequate sampling plan for field researchers obtaining microbial biomass for 16S rRNA gene sequencing, applicable specifically to low biomass oil and gas-producing environments. Forty-nine different samples were collected by filtering specific volumes of produced water from a hydraulically fractured well producing from the Niobrara Shale. Water was collected in two different sampling events 24 h apart. Four to five samples were collected from 11 specific volumes. These samples along with eight different blanks were submitted for analysis. DNA was extracted from each sample, and quantitative polymerase chain reaction (qPCR) and 16S rRNA Illumina MiSeq gene sequencing were performed to determine relative concentrations of biomass and microbial community composition, respectively. The qPCR results varied across sampled volumes, while no discernible trend correlated contamination to volume of water filtered. This suggests that collecting a larger volume of sample may not result in larger biomass concentrations or better representation of a sampled environment. Researchers could prioritize collecting many low volume samples over few high-volume samples. Our results suggest that there also may be variability in the concentration of microbial communities present in produced waters over short (i.e., hours) time scales, which warrants further investigation. Submission of multiple blanks is also vital to determining how contamination or low biomass effects may influence a sample set collected from an unknown environment.

从深部地下环境中获取微生物DNA并开展测序，面临诸多复杂难题，从样本污染到实验结果无法重复均属此类。这类环境因具备刺激微生物产甲烷的潜力而备受学界关注，但从中获取的多数样本生物量极低。因此，此类环境样本的研究难度极大，测序结果极易受污染影响：样本中极低的生物量可能被污染DNA完全掩盖，进而产生误导性实验结果。此外，在这类环境开展野外工作困难重重，研究人员往往难以进入现场且现场作业时间有限。因此，理想的采样方案应在短时间内采集尽可能多样本的同时，确保获得最优实验结果。本研究旨在为开展微生物生物量采样、用于16S rRNA基因测序（16S rRNA gene sequencing）的野外研究者，提供适配低生物量油气生产环境的可行采样方案建议。研究人员从一口开采尼奥布拉拉组页岩（Niobrara Shale）的水力压裂井中，通过过滤不同体积的采出水采集了49份不同样本，两次采样事件间隔24小时。针对11种特定体积的水样，各采集4至5份重复样本；连同8份空白对照样本一并送检分析。对每份样本提取DNA后，分别采用定量聚合酶链反应（quantitative polymerase chain reaction, qPCR）与Illumina MiSeq平台的16S rRNA基因测序，以测定生物量相对浓度与微生物群落组成。qPCR结果随采样体积的不同存在差异，但未观察到污染程度与过滤水样体积存在明确相关趋势。这表明采集更大体积的样本，未必能获得更高的生物量浓度，或是更具代表性的采样环境特征。研究人员可优先采集大量小体积样本，而非少量大体积样本。我们的研究结果还显示，采水中的微生物群落浓度在短时间尺度（即数小时）内或存在波动，这有待进一步探究。此外，提交多份空白对照样本，对于明确污染或低生物量如何影响未知环境采集的样本集至关重要。