Abstract
Methods for the study of member species in complex microbial communities remain a high priority, particularly for rare and/or novel member species that might play an important ecological role. Specifically, methods that link genomic information of member species with its spatial structure are lacking. This study adopts an integrative workflow that permits the characterisation of previously unclassified bacterial taxa from microbiomes through: (1) imaging of the spatial structure; (2) taxonomic classification and (3) genome recovery. Our study attempts to bridge the gaps between metagenomics/metatranscriptomics and high-resolution biomass imaging methods by developing new fluorescence in situ hybridisation (FISH) probes—termed as R-Probes—from shotgun reads that harbour hypervariable regions of the 16S rRNA gene. The sample-centric design of R-Probes means that probes can directly hybridise to OTUs as detected in shotgun sequencing surveys. The primer-free probe design captures larger microbial diversity as compared to canonical probes. R-Probes were designed from deep-sequenced RNA-Seq datasets for both FISH imaging and FISH–Fluorescence activated cell sorting (FISH–FACS). FISH–FACS was used for target enrichment of previously unclassified bacterial taxa prior to downstream multiple displacement amplification (MDA), genomic sequencing and genome recovery. After validation of the workflow on an axenic isolate of Thauera species, the techniques were applied to investigate two previously uncharacterised taxa from a tropical full-scale activated sludge community. In some instances, probe design on the hypervariable region allowed differentiation to the species level. Collectively, the workflow can be readily applied to microbiomes for which shotgun nucleic acid survey data is available.
Highlights
The study of complex microbial communities typically involves a combination of analyses needed to gain a comprehensive understanding of their composition, structure and function.[1]
In this study, existing gaps between whole community shotgun sequencing and high-resolution biomass imaging methods were bridged by taking advantage of fluorescence in situ hybridisation (FISH) probes designed directly from 16S rRNA gene variable regions obtained from shotgun sequencing data
While FISH probes have previously been designed from the V6 region of 16S rRNA gene from pyrosequences,[14] the motivation for the design of R-Probes is to mitigate bias that may be introduced during PCR amplification of the hypervariable region through the extraction of taxonomically informative tag sequences directly from shotgun sequencing data.[16]
Summary
The study of complex microbial communities typically involves a combination of analyses needed to gain a comprehensive understanding of their composition, structure and function.[1]. Given the large-scale deployment of high-throughput shotgun sequencing for microbial community profiling, the resulting short length reads derived from hypervariable regions of the SSU gene present an opportunity for the design of FISH probes, as demonstrated by Hasegawa et al.[14] using 16S rRNA gene amplicon sequences. This was performed to attain stringent from taxonomically informative tag sequences (33 bp) from the hypervariable region (V4–V7) of the 16S rRNA as defined by our previously developed RiboTagger software.[16] The RiboTagger softhybridisation at a standardised hybridisation temperature of 46 °C.19. Following validation studies using axenic cultures, the method was applied to study two previously uncharacterised microbial taxa (not represented in the SILVA SSU17 and the NCBI Genome database18) from an activated sludge community sampled from a full-scale wastewater plant. Probe name as described by Alm et al.,[20] followed by an underscore
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