Metagenomic Quantification of Genes with Internal Standards.
Metagenomic Quantification of Genes with Internal Standards.
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具有内部标准标准的基因的宏基因组量化。
DOI：
10.1128/mbio.03173-20
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发表时间：
2021-02-02
期刊：
影响因子：
6.4
通讯作者：
Wigginton K
中科院分区：
文献类型：
--
作者：
Crossette E;Gumm J;Langenfeld K;Raskin L;Duhaime M;Wigginton K
定量聚合酶链式反应和元基因组学是几十年来一直提供对生物过程的洞察的核心分子技术，从监测空间和时间基因动态到跟踪ARGs或病原体。仍然需要一种工具，可以将样本中数千个相关基因量化为每个样本量或体积的基因副本。我们证明了一种装配无关和便于插入的元基因组量化方法可以用于同时筛选和量化超过2,000个基因，同时提供与定量PCR(QPCR)相当的绝对基因浓度。从牛粪泥、消化液和堆肥中提取的DNA被添加到一种海洋细菌的基因组DNA中，并使用Illumina HiSeq4000进行了测序。我们比较了五个抗菌素耐药基因(ARGs)的基因拷贝浓度，以每质量样本的基因拷贝数为单位，这些基因(ARGs)由(I)我们的定量元基因组学方法、(Ii)靶向qPCR方法和(Iii)涉及元基因组学和基于qPCR的16S rRNA基因定量的混合量化方法产生。虽然qPCR达到了较低的定量极限，但元基因组学方法避免了qPCR方法固有的引物专一性引起的偏差，并且能够检测到比qPCR分析多几个数量级的基因。我们使用该方法同时量化了综合耐药性数据库(CARD)中的ARGs。我们观察到，三个养殖场的粪便处理不同阶段，四环素抗性基因的总丰度是一致的，但不同的样品由不同的四环素抗性基因家族主导。
qPCR and metagenomics are central molecular techniques that have offered insights into biological processes for decades, from monitoring spatial and temporal gene dynamics to tracking ARGs or pathogens. Still needed is a tool that can quantify thousands of relevant genes in a sample as gene copies per sample mass or volume. We demonstrate that an assembly-independent and spike-in facilitated metagenomic quantification approach can be used to screen and quantify over 2,000 genes simultaneously, while delivering absolute gene concentrations comparable to those for quantitative PCR (qPCR). DNA extracted from dairy manure slurry, digestate, and compost was spiked with genomic DNA from a marine bacterium and sequenced using the Illumina HiSeq4000. We compared gene copy concentrations, in gene copies per mass of sample, of five antimicrobial resistance genes (ARGs) generated with (i) our quantitative metagenomic approach, (ii) targeted qPCR, and (iii) a hybrid quantification approach involving metagenomics and qPCR-based 16S rRNA gene quantification. Although qPCR achieved lower quantification limits, the metagenomic method avoided biases caused by primer specificity inherent to qPCR-based methods and was able to detect orders of magnitude more genes than is possible with qPCR assays. We used the approach to simultaneously quantify ARGs in the Comprehensive Antimicrobial Resistance Database (CARD). We observed that the total abundance of tetracycline resistance genes was consistent across different stages of manure treatment on three farms, but different samples were dominated by different tetracycline resistance gene families.