A pipeline for targeted metagenomics of environmental bacteria

Anissa Grieb,Danielle Goudeau,Janey Lee,Bernhard M Fuchs,Robert M Bowers,Monike Oggerin,Tanja Woyke,Rex R Malmstrom

doi:10.1186/s40168-020-0790-7

Abstract

BackgroundMetagenomics 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.MethodsCells 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.ResultsPure 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.ConclusionsWith 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.Brr92jERKGjhdPpcgRJbTxVideo abstract.

Highlights

Metagenomics and single cell genomics provide a window into the genetic repertoire of yet uncultivated microorganisms, but both methods are usually taxonomically untargeted
Quantification of the hybridization chain reaction (HCR)-fluorescence in situ hybridization (FISH) signal intensity depending on cell fixation We tested nine different fixation methods on four different bacterial strains to determine the impact of fixation on the fluorescence labeling of the cells, and quantified the fluorescence intensities after HCR-FISH by flow cytometry (Fig. 1)
We have successfully developed a pipeline for the targeted enrichment of uncultivated bacterial clades based on HCR-FISH and fluorescence activated cell sorting (FACS), enabling subsequent whole genome sequencing and the retrieval of metagenome assembled genomes (MAGs) for genomic annotation and characterization

Summary

Introduction

Metagenomics and single cell genomics provide a window into the genetic repertoire of yet uncultivated microorganisms, but both methods are usually taxonomically untargeted. The resulting single amplified genomes (SAGs) are generally incomplete [7, 8]. Both metagenomics and SCG are usually untargeted and this random selection might challenge access to rare organisms [9, 10]. These could be recovered by a targeted approach where specific microbial groups are enriched prior to sequencing

Methods

Results

Discussion

Conclusion