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Abstract Exploring the diversity of diazotrophs is key to understanding their role in supplying fixed nitrogen that supports marine productivity. A nested PCR assay using the universal primer set nifH1-nifH4, which targets the nitrogenase (nifH) gene, is a widely used approach for studying marine diazotrophs by amplicon sequencing. Metagenomics, direct sequencing of DNA without PCR, has provided complementary views of the diversity of marine diazotrophs. A significant fraction of the metagenome-derived nifH sequences (e.g. Planctomycete- and Proteobacteria-affiliated) were reported to have nucleotide mismatches with the nifH1-nifH4 primers, leading to the suggestion that nifH amplicon sequencing does not detect specific diazotrophic taxa and underrepresents diazotroph diversity. Here, we report that these mismatches are mostly located in a single-base at the 5′-end of the nifH4 primer, which does not impact detection of the nifH genes. This is demonstrated by the presence of nifH genes that contain the nucleotide mismatches in a recent compilation of global ocean nifH amplicon datasets, with high relative abundances detected in a variety of samples. While the metagenome- and metatranscriptome-derived nifH genes accounted for 4.4% of the total amplicon sequence variants from the global ocean nifH amplicon database, the corresponding amplicon sequence variants can have high relative abundances (accounting for 47% of the reads in the database). These analyses underscore that nifH amplicon sequencing using the nifH1-nifH4 primers is an important tool for studying diversity of marine diazotrophs, particularly as a complement to metagenomics which can provide taxonomic and metabolic information for some dominant groups.
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vAMPirus : A versatile amplicon processing and analysis program for studying viruses
Abstract Amplicon sequencing is an effective and increasingly applied method for studying viral communities in the environment. Here, we present vAMPirus, a user‐friendly, comprehensive, and versatile DNA and RNA virus amplicon sequence analysis program, designed to support investigators in exploring virus amplicon sequencing data and running informed, reproducible analyses. vAMPirus intakes raw virus amplicon libraries and, by default, performs nucleotide‐ and amino acid‐based analyses to produce results such as sequence abundance information, taxonomic classifications, phylogenies and community diversity metrics. The vAMPirus analytical framework leverages 16 different opensource tools and provides optional approaches that can increase the ratio of biological signal‐to‐noise and thereby reveal patterns that would have otherwise been masked. Here, we validate the vAMPirus analytical framework and illustrate its implementation as a general virus amplicon sequencing workflow by recapitulating findings from two previously published double‐stranded DNA virus datasets. As a case study, we also apply the program to explore the diversity and distribution of a coral reef‐associated RNA virus. vAMPirus is streamlined within Nextflow, offering straightforward scalability, standardization and communication of virus lineage‐specific analyses. The vAMPirus framework is designed to be adaptable; community‐driven analytical standards will continue to be incorporated as the field advances. vAMPirus supports researchers in revealing patterns of virus diversity and population dynamics in nature, while promoting study reproducibility and comparability.
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- PAR ID:
- 10566358
- Publisher / Repository:
- Wiley
- Date Published:
- Journal Name:
- Molecular Ecology Resources
- Volume:
- 24
- Issue:
- 6
- ISSN:
- 1755-098X
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript
- Journal Article:
- https://doi.org/10.1111/1755-0998.13978
