Evaluation of the Ion Torrent Personal Genome Machine for Gene-Targeted Studies Using Amplicons of the Nitrogenase Gene
            <i>nifH</i>

Zhang, Bangzhou; Penton, C. Ryan; Xue, Cong; Wang, Qiong; Zheng, Tao; Tiedje, James M.

doi:10.1128/aem.00111-15

Cited by 27 publications

(21 citation statements)

References 34 publications

(39 reference statements)

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“…In comparison to the FunGene tool, TaxADivA meets particular needs, such as filtering of cluster IV/V sequences (see below) and trimming of highly degenerate primer sequences. Trimming of primer sequences may fail during quality control (33), likely due to the extreme degeneracy of nifH primers, and thus we adapted the primer trimming function in the TaxADivA script to trim a specified number of bases off the flanking ends of the amplicon.…”

Section: Discussionmentioning

confidence: 99%

Diazotroph Community Characterization via a High-Throughput nifH Amplicon Sequencing and Analysis Pipeline

Gaby

Rishishwar

Valderrama-Aguirre

et al. 2018

Appl Environ Microbiol

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The dinitrogenase reductase gene () is the most widely established molecular marker for the study of nitrogen-fixing prokaryotes in nature. A large number of PCR primer sets have been developed for amplification, and the effective deployment of these approaches should be guided by a rapid, easy-to-use analysis protocol. Bioinformatic analysis of marker gene sequences also requires considerable expertise. In this study, we advance the state of the art for analysis by evaluating primer set performance, developing an improved amplicon sequencing workflow, and implementing a user-friendly bioinformatics pipeline. The developed amplicon sequencing workflow is a three-stage PCR-based approach that uses established technologies for incorporating sample-specific barcode sequences and sequencing adapters. Based on our primer evaluation, we recommend the Ando primer set be used with a modified annealing temperature of 58°C, as this approach captured the largest diversity of templates, including paralog cluster IV/V sequences. To improve sequence analysis, we developed a computational pipeline which infers taxonomy and optionally filters out paralog sequences. In addition, we employed an empirical model to derive optimal operational taxonomic unit (OTU) cutoffs for the gene at the species, genus, and family levels. A comprehensive workflow script named TaxADivA (TAXonomy Assignment and DIVersity Assessment) is provided to ease processing and analysis of amplicons. Our approach is then validated through characterization of diazotroph communities across environmental gradients in beach sands impacted by the Deepwater Horizon oil spill in the Gulf of Mexico, in a peat moss-dominated wetland, and in various plant compartments of a sugarcane field. Nitrogen availability often limits ecosystem productivity, and nitrogen fixation, exclusive to prokaryotes, comprises a major source of nitrogen input that sustains food webs. The gene, which codes for the iron protein of the nitrogenase enzyme, is the most widely established molecular marker for the study of nitrogen-fixing microorganisms (diazotrophs) in nature. In this study, a flexible sequencing/analysis pipeline, named TaxADivA, was developed for amplicons produced by Illumina paired-end sequencing, and it enables an inference of taxonomy, performs clustering, and produces output in formats that may be used by programs that facilitate data exploration and analysis. Diazotroph diversity and community composition are linked to ecosystem functioning, and our results advance the phylogenetic characterization of diazotroph communities by providing empirically derived similarity cutoffs for species, genus, and family levels. The utility of our pipeline is validated for diazotroph communities in a variety of ecosystems, including contaminated beach sands, peatland ecosystems, living plant tissues, and rhizosphere soil.

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Section: Discussionmentioning

confidence: 99%

Diazotroph Community Characterization via a High-Throughput nifH Amplicon Sequencing and Analysis Pipeline

Gaby

Rishishwar

Valderrama-Aguirre

et al. 2018

Appl Environ Microbiol

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“…PEPPA thus supports an analysis of the accumulation of pseudogenes with time by individual bacterial lineages, and might be useful for studies concerning the importance of pseudogenes for potential adaptations to new environments. Draft genome assemblies based on 454 or IonTorrent sequencing include elevated numbers of single-base insertions and deletions due to inaccurate sequencing (Shao et al 2013;Zhang et al 2015). Including such genomes in an analysis reduces the quality of the pan-genome for all state of the art pipelines.…”

Section: Effects Of Internal Annotations By Peppa Our Analyses Of 15mentioning

confidence: 99%

Accurate reconstruction of bacterial pan- and core- genomes with PEPPAN

Zhou

Charlesworth

Achtman

2020

Preprint

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Bacterial genomes have been shaped by a complex evolutionary history, including extensive homologous recombination, horizontal gene transfer, gene loss, and gene duplications. A pan-genome, consisting of all genes within a defined set of bacterial genomes, can provide the basis for phylogenetic inferences and population studies.Here we introduce PEPPA, a pipeline that can construct a pan-genome from thousands of genetically diversified bacterial genomes. PEPPA implements a combination of treeand synteny-based approaches to identify and exclude paralogous genes, which facilitates the ready construction of schemes for whole genome multi-locus sequence typing (wgMLST) and core genome MLST. PEPPA also implements similarity-based gene predictions that support consistent annotations of genes and pseudogenes in individual genomes, and which are nearly as accurate as expert manual curation. The PEPPA package includes PEPPA_parser, which calculates trees based on accessory gene content and MLST core genome allelic differences. We also present SimPan, a novel pipeline for simulating the evolution of a bacterial pan-genome. PEPPA was compared with four state-of-the-art pan-genome pipelines on both empirical and simulated datasets. It demonstrated greater accuracy and specificity than any other pipeline, and was almost as fast as any of them at calculating a pan-genome. To demonstrate its abilities, PEPPA was used to construct a Streptococcus pan-genome of >40,000 genes from 3,170 representative genomes spanning at least 80 species. The resulting gene and allelic trees provide an unprecedented overview of the genomic diversity of this genus. Kilian M, Poulsen K, Blomqvist T, Havarstein LS, Bek-Thomsen M, Tettelin H, Sorensen UB. 2008. Evolution of Streptococcus pneumoniae and its close commensal relatives. PLoS ONE 3: e2683. Kilian M and Tettelin H. 2019. Identification of virulence-associated properties by comparative genome analysis of Streptococcus pneumoniae, S. pseudopneumoniae, S. mitis, three S. oralis subspecies, and S. infantis. MBio 10. Konstantinidis KT, Rossello-Mora R, Amann R. 2017. Uncultivated microbes in need of their own taxonomy. ISME J 11: 2399-2406. Koonin EV. 2016. Splendor and misery of adaptation, or the importance of neutral null for understanding evolution. BMC Biol 14: 114.

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“…Current NGS platforms include GS FLX Titanium/GS FLX Junior from Roche, SOLiD and Ion Torrent platforms from Life Sciences, the PacBio RS II from Pacific Biosciences, and Genome Analyzer/HiSeq/MiSeq/NextSeq from Illumina. Owing to the specifics of enzymology, chemistry, high-resolution optics, hardware, and software, each platform differs in the types of sequencing errors (i.e., artifacts) (reviewed in [ 33 – 35 ]) and quantity of data produced (for detailed methodology, see [ 1 , 3 – 5 , 36 – 44 ]). Most NGS projects sequence genomes via the assembly of short (60–150 base pair or bp) reads.…”

Section: Introductionmentioning

confidence: 99%

Next-generation genotyping of hypervariable loci in many individuals of a non-model species: technical and theoretical implications

et al. 2016

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BackgroundAcross species, diversity at the Major Histocompatibility Complex (MHC) is critical to disease resistance and population health; however, use of MHC diversity to quantify the genetic health of populations has been hampered by the extreme variation found in MHC genes. Next generation sequencing (NGS) technology generates sufficient data to genotype even the most diverse species, but workflows for distinguishing artifacts from alleles are still under development. We used NGS to evaluate the MHC diversity of over 300 captive and wild ring-tailed lemurs (Lemur catta: Primates: Mammalia). We modified a published workflow to address errors that arise from deep sequencing individuals and tested for evidence of selection at the most diverse MHC genes.ResultsIn addition to evaluating the accuracy of 454 Titanium and Ion Torrent PGM for genotyping large populations at hypervariable genes, we suggested modifications to improve current methods of allele calling. Using these modifications, we genotyped 302 out of 319 individuals, obtaining an average sequencing depth of over 1000 reads per amplicon. We identified 55 MHC-DRB alleles, 51 of which were previously undescribed, and provide the first sequences of five additional MHC genes: DOA, DOB, DPA, DQA, and DRA. The additional five MHC genes had one or two alleles each with little sequence variation; however, the 55 MHC-DRB alleles showed a high dN/dS ratio and trans-species polymorphism, indicating a history of positive selection. Because each individual possessed 1–7 MHC-DRB alleles, we suggest that ring-tailed lemurs have four, putatively functional, MHC-DRB copies.ConclusionsIn the future, accurate genotyping methods for NGS data will be critical to assessing genetic variation in non-model species. We recommend that future NGS studies increase the proportion of replicated samples, both within and across platforms, particularly for hypervariable genes like the MHC. Quantifying MHC diversity within non-model species is the first step to assessing the relationship of genetic diversity at functional loci to individual fitness and population viability. Owing to MHC-DRB diversity and copy number, ring-tailed lemurs may serve as an ideal model for estimating the interaction between genetic diversity, fitness, and environment, especially regarding endangered species.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2503-y) contains supplementary material, which is available to authorized users.

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Evaluation of the Ion Torrent Personal Genome Machine for Gene-Targeted Studies Using Amplicons of the Nitrogenase Gene nifH

Cited by 27 publications

References 34 publications

Diazotroph Community Characterization via a High-Throughput nifH Amplicon Sequencing and Analysis Pipeline

Diazotroph Community Characterization via a High-Throughput nifH Amplicon Sequencing and Analysis Pipeline

Accurate reconstruction of bacterial pan- and core- genomes with PEPPAN

Next-generation genotyping of hypervariable loci in many individuals of a non-model species: technical and theoretical implications

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