Evaluation of Primers Targeting the Diazotroph Functional Gene and Development of NifMAP - A Bioinformatics Pipeline for Analyzing nifH Amplicon Data

Frontiers in Microbiology
Roey AngelDagmar Woebken

Abstract

Diazotrophic microorganisms introduce biologically available nitrogen (N) to the global N cycle through the activity of the nitrogenase enzyme. The genetically conserved dinitrogenase reductase (nifH) gene is phylogenetically distributed across four clusters (I-IV) and is widely used as a marker gene for N2 fixation, permitting investigators to study the genetic diversity of diazotrophs in nature and target potential participants in N2 fixation. To date there have been limited, standardized pipelines for analyzing the nifH functional gene, which is in stark contrast to the 16S rRNA gene. Here we present a bioinformatics pipeline for processing nifH amplicon datasets - NifMAP ("NifH MiSeq Illumina Amplicon Analysis Pipeline"), which as a novel aspect uses Hidden-Markov Models to filter out homologous genes to nifH. By using this pipeline, we evaluated the broadly inclusive primer pairs (Ueda19F-R6, IGK3-DVV, and F2-R6) that target the nifH gene. To evaluate any systematic biases, the nifH gene was amplified with the aforementioned primer pairs in a diverse collection of environmental samples (soils, rhizosphere and roots samples, biological soil crusts and estuarine samples), in addition to a nifH mock community consisting of si...Continue Reading

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Citations

Nov 12, 2019·Frontiers in Plant Science·Rares LucaciuThomas Rattei
Dec 25, 2019·Microbial Ecology·Amisha T Poret-PetersonDaniel A Kluepfel
Nov 22, 2019·Frontiers in Molecular Biosciences·Vadakattu V S R GuptaJames M Tiedje
Oct 11, 2020·FEMS Microbiology Ecology·Darian N SmercinaLisa K Tiemann
Feb 14, 2021·Bioinformatics·Bennett J Kapili, Anne E Dekas
Sep 3, 2021·Molecular Ecology·Valentine MeunierFanny Houlbrèque
Dec 4, 2021·The ISME Journal·Nils RädeckerChristian R Voolstra
Jan 14, 2022·GigaScience·Christophe DjemielLionel Ranjard

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Datasets Mentioned

BETA
M20568
PRJNA4
32667

Methods Mentioned

BETA
PCR
Illumina sequencing
electrophoresis
amplicon sequencing
Assay
PCRs

Software Mentioned

BLAST
HMMER
MEGAN
RAxML
BLASTP
MAFFT L - INS - i
EPA
MAFFT
FrameBot
NifH MiSeq Illumina Amplicon Analysis Pipeline

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