An optimized approach for processing of frozen lung and lavage samples for microbiome studies.
An optimized approach for processing of frozen lung and lavage samples for microbiome studies.
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DOI：
10.1371/journal.pone.0265891
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发表时间：
2022
期刊：
影响因子：
3.7
通讯作者：
Gonzalez-Juarbe N
中科院分区：
文献类型：
--
作者：
Wiscovitch-Russo R;Singh H;Oldfield LM;Fedulov AV;Gonzalez-Juarbe N
呼吸道具有生物量低且多样性有限的常驻微生物组。由于细菌与宿主DNA的比例不均匀，这导致测序样品制备困难，特别是对于小组织样品，如小鼠肺。我们比较了目前用于微生物组研究中DNA提取的程序的有效性。收集支气管肺泡灌洗液（BALF）和肺组织样本，测试不同形式的样本预处理和提取方法，以提高细菌DNA产量并优化文库制备。使用机械裂解（肺组织）和一步离心（BALF）的预处理方法提取DNA增加了DNA产量和样品的细菌含量。相比之下，酚氯仿异戊醇（PCI）提取和巢式PCR检测后，环境污染显着增加。虽然PCI是微生物组研究中使用的标准程序，但我们的数据表明，它对冷冻低生物量样品的DNA提取效率不高。最后，测试了DNA富集试剂盒，发现其提高了肺组织的16S拷贝数，微生物组成略有变化。总的来说，我们提出了一种标准化的方法，以提供高产量的DNA，并提高低微生物生物量冷冻样品的测序覆盖率，使污染最小。
The respiratory tract has a resident microbiome with low biomass and limited diversity. This results in difficulties with sample preparation for sequencing due to uneven bacteria-to-host DNA ratio, especially for small tissue samples such as mouse lungs. We compared effectiveness of current procedures used for DNA extraction in microbiome studies. Bronchoalveolar lavage fluid (BALF) and lung tissue samples were collected to test different forms of sample pre-treatment and extraction methods to increase bacterial DNA yield and optimize library preparation. DNA extraction using a pre-treatment method of mechanical lysis (lung tissue) and one-step centrifugation (BALF) increased DNA yield and bacterial content of samples. In contrast, a significant increase of environmental contamination was detected after phenol chloroform isoamyl alcohol (PCI) extraction and nested PCR. While PCI has been a standard procedure used in microbiome studies, our data suggests that it is not efficient for DNA extraction of frozen low biomass samples. Finally, a DNA Enrichment kit was tested and found to improve the 16S copy number of lung tissue with a minor shift in microbial composition. Overall, we present a standardized method to provide high yielding DNA and improve sequencing coverage of low microbial biomass frozen samples with minimal contamination.
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DOI：
10.1016/j.cgh.2018.09.017
发表时间：
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期刊：
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