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A pipeline for targeted metagenomics of environmental bacteria.
Microbiome ( IF 15.5 ) Pub Date : 2020-02-15 , DOI: 10.1186/s40168-020-0790-7
Anissa Grieb 1 , Robert M Bowers 2 , Monike Oggerin 1 , Danielle Goudeau 2 , Janey Lee 2 , Rex R Malmstrom 2 , Tanja Woyke 2 , Bernhard M Fuchs 1
Affiliation  

BACKGROUND Metagenomics and single cell genomics provide a window into the genetic repertoire of yet uncultivated microorganisms, but both methods are usually taxonomically untargeted. The combination of fluorescence in situ hybridization (FISH) and fluorescence activated cell sorting (FACS) has the potential to enrich taxonomically well-defined clades for genomic analyses. METHODS Cells hybridized with a taxon-specific FISH probe are enriched based on their fluorescence signal via flow cytometric cell sorting. A recently developed FISH procedure, the hybridization chain reaction (HCR)-FISH, provides the high signal intensities required for flow cytometric sorting while maintaining the integrity of the cellular DNA for subsequent genome sequencing. Sorted cells are subjected to shotgun sequencing, resulting in targeted metagenomes of low diversity. RESULTS Pure cultures of different taxonomic groups were used to (1) adapt and optimize the HCR-FISH protocol and (2) assess the effects of various cell fixation methods on both the signal intensity for cell sorting and the quality of subsequent genome amplification and sequencing. Best results were obtained for ethanol-fixed cells in terms of both HCR-FISH signal intensity and genome assembly quality. Our newly developed pipeline was successfully applied to a marine plankton sample from the North Sea yielding good quality metagenome assembled genomes from a yet uncultivated flavobacterial clade. CONCLUSIONS With the developed pipeline, targeted metagenomes at various taxonomic levels can be efficiently retrieved from environmental samples. The resulting metagenome assembled genomes allow for the description of yet uncharacterized microbial clades. Video abstract.

中文翻译:

针对环境细菌的宏基因组学的管道。

背景技术元基因组学和单细胞基因组学为了解尚未培养的微生物的遗传库提供了一个窗口,但是这两种方法通常在分类学上都没有针对性。荧光原位杂交(FISH)和荧光激活细胞分选(FACS)的结合具有丰富分类学上定义明确的进化枝进行基因组分析的潜力。方法通过流式细胞仪分选,基于分类信号的FISH探针杂交的细胞得以富集。最近开发的FISH程序,杂交链反应(HCR)-FISH,提供了流式细胞仪分选所需的高信号强度,同时保持了细胞DNA的完整性,可用于后续的基因组测序。排序后的细胞经过散弹枪测序,导致靶向性低的元基因组。结果使用不同分类学组的纯培养物来(1)适应和优化HCR-FISH方案,(2)评估各种细胞固定方法对细胞分类的信号强度以及后续基因组扩增和测序质量的影响。就HCR-FISH信号强度和基因组组装质量而言,乙醇固定细胞获得了最佳结果。我们新开发的管道已成功应用于北海的一个海洋浮游生物样品,该样品从尚未培养的黄细菌进化枝中产生了高质量的基因组组装基因组。结论利用开发的管线,可以从环境样品中有效地检索各种分类学水平的靶向元基因组。所得的由基因组组装的基因组可用于描述尚未鉴定的微生物进化枝。录像摘要。
更新日期:2020-04-22
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