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Abstract 16S rRNA targeted amplicon sequencing is an established standard for elucidating microbial community composition. While high‐throughput short‐read sequencing can elicit only a portion of the 16S rRNA gene due to their limited read length, third generation sequencing can read the 16S rRNA gene in its entirety and thus provide more precise taxonomic classification. Here, we present a protocol for generating full‐length 16S rRNA sequences with Oxford Nanopore Technologies (ONT) and a microbial community profile with Emu. We select Emu for analyzing ONT sequences as it leverages information from the entire community to overcome errors due to incomplete reference databases and hardware limitations to ultimately obtain species‐level resolution. This pipeline provides a low‐cost solution for characterizing microbiome composition by exploiting real‐time, long‐read ONT sequencing and tailored software for accurate characterization of microbial communities. © 2024 Wiley Periodicals LLC. Basic Protocol: Microbial community profiling with Emu Support Protocol 1: Full‐length 16S rRNA microbial sequences with Oxford Nanopore Technologies sequencing platform Support Protocol 2: Building a custom reference database for Emu
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Towards quantitative microbiome community profiling using internal standards
An inherent issue in high-throughput rRNA gene tag sequencing microbiome surveys is that they provide compositional data in relative abundances. This often leads to spurious correlations, making the interpretation of relationships to biogeochemical rates challenging. To overcome this issue, we quantitatively estimated the abundance of microorganisms by spiking in known amounts of internal DNA standards. Using a 3-year sample set of diverse microbial communities from the Western Antarctica Peninsula, we demonstrated that the internal standard method yielded community profiles and taxon cooccurrence patterns substantially different from those derived using relative abundances. We found that the method provided results consistent with the traditional CHEMTAX analysis of pigments and total bacterial counts by flow cytometry. Using the internal standard method, we also showed that chloroplast 16S rRNA gene data in microbial surveys can be used to estimate abundances of certain eukaryotic phototrophs such as cryptophytes and diatoms. In Phaeocystis, scatter in the 16S/18S rRNA gene ratio may be explained by physiological adaptation to environmental conditions. We conclude that the internal standard method, when applied to rRNA gene microbial community profiling, is quantitative and that its application will substantially improve our understanding of microbial ecosystems.
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- Award ID(s):
- 1643534
- PAR ID:
- 10091386
- Date Published:
- Journal Name:
- Applied and environmental microbiology
- ISSN:
- 1098-5336
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/http://dx.doi.org/10.1128/AEM.02634-18
