Metagenome complexity and template length are the main causes of bias in PCR‐based bacteria community analysis

Peng, Wentao; Li, Xiangmin; Wang, Chuang; Cao, Hui; Cui, Zhongli

doi:10.1002/jobm.201800265

Cited by 16 publications

(9 citation statements)

References 37 publications

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“…Importantly, primer amplification bias goes beyond entirely missing certain clades. GϩC-rich templates might perform differently than those rich in AϩT (26), and taxa with longer variants might not be detected as efficiently as others with a shorter variable region (27). These biases are compounded by the exponential nature of PCR amplification.…”

Section: Resultsmentioning

confidence: 99%

Assessment of In Vitro and In Silico Protocols for Sequence-Based Characterization of the Human Vaginal Microbiome

Hugerth

Pereira

Zha

et al. 2020

mSphere

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The vaginal microbiome has been connected to a wide range of health outcomes. This has led to a thriving research environment but also to the use of conflicting methodologies to study its microbial composition. Here, we systematically assessed best practices for the sequencing-based characterization of the human vaginal microbiome. As far as 16S rRNA gene sequencing is concerned, the V1-V3 region performed best in silico, but limitations of current sequencing technologies meant that the V3-V4 region performed equally well. Both approaches presented very good agreement with qPCR quantification of key taxa, provided that an appropriate bioinformatic pipeline was used. Shotgun metagenomic sequencing presents an interesting alternative to 16S rRNA gene amplification and sequencing but requires deeper sequencing and more bioinformatic expertise and infrastructure. We assessed different tools for the removal of host reads and the taxonomic annotation of metagenomic reads, including a new, easy-to-build and -use reference database of vaginal taxa. This curated database performed as well as the best-performing previously published strategies. Despite the many advantages of shotgun sequencing, none of the shotgun approaches assessed here agreed with the qPCR data as well as the 16S rRNA gene sequencing. IMPORTANCE The vaginal microbiome has been connected to various aspects of host health, including susceptibility to sexually transmitted infections as well as gynecological cancers and pregnancy outcomes. This has led to a thriving research environment but also to conflicting available methodologies, including many studies that do not report their molecular biological and bioinformatic methods in sufficient detail to be considered reproducible. This can lead to conflicting messages and delay progress from descriptive to intervention studies. By systematically assessing best practices for the characterization of the human vaginal microbiome, this study will enable past studies to be assessed more critically and assist future studies in the selection of appropriate methods for their specific research questions.

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Section: Resultsmentioning

confidence: 99%

Assessment of In Vitro and In Silico Protocols for Sequence-Based Characterization of the Human Vaginal Microbiome

Hugerth

Pereira

Zha

et al. 2020

mSphere

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“…Polymerase chain reaction (PCR), a technique used to make numerous copies of a specific segment of DNA quickly and accurately. However, PCR usually produces bias, resulting in inaccurate experimental results because of lack of perfect working primers for many of the gene families involved (Peng et al, 2018). In the present study, shotgun metagenome sequencing technology was used to investigate the complete sulfur cycle in mangroves.…”

Section: Discussionmentioning

confidence: 99%

Impacts ofDesulfobacteralesandChromatialeson sulfate reduction in the subtropical mangrove ecosystem as revealed by SMDB analysis

et al. 2020

Preprint

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Motivation Sulfate reduction is an important process in the sulfur cycle. However, the relationship between this process and the genotype of microorganisms involved in subtropical mangrove ecosystems is poorly understood. Genotyping can identify and is crucial for sulfate reduction in mangrove ecosystems with high sulfur concentrations. A professional and efficient gene integration database of sulfur metabolism has not been established yet. Results This work aimed to evaluate sulfate reduction by microorganisms in mangroves by using a sulfur metabolism gene integrative database (SMDB) that was mainly constructed to analyze the sulfur cycle (sub) gene families quickly and accurately. The database achieved high coverage, fast retrieval, and low false positives. Relative enrichment of sulfate adenylyltransferase indicated that environmental factors select for a partial dissimilatory sulfate reduction process. Furthermore, the sulfate reduction community compensates by producing certain sulfate-reduction genes in response to high-sulfur environments in mangrove sediments. Taxonomic assignment of dissimilatory sulfate-reduction genes revealed that Crenarchaeota and Halobacterota are completely responsible for this process. Sulfite reductase can help the community cope with the toxic sulfite produced by these Archaea phyla. Collectively, these findings suggested that Halobacterota and Crenarchaeota play essential roles in dissimilatory sulfate reduction. Availability and implementation SMDB is freely available under http://smdb.gxu.edu.cn/ and https://github.com/taylor19891213/sulfur-metabolism-gene-database. Supplementary information Supplementary data are available at Bioinformatics online.

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“…In order to achieve accurate sequencing results, many factors have to be considered when designing a sequencing study. Among the processing steps that affects metagenomic analyses, PCR-based strategies are source of biases because of differential amplification efficiency among templates in terms of target length, primer binding sites and GC content ( 11 – 13 ). Many studies demonstrate potential amplification biases that are introduced with the use of various commonly utilized primers.…”

Section: Introductionmentioning

confidence: 99%

MinION Sequencing of Yeast Mock Communities To Assess the Effect of Databases and ITS-LSU Markers on the Reliability of Metabarcoding Analysis

et al. 2023

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Metagenome complexity and template length are the main causes of bias in PCR‐based bacteria community analysis

Cited by 16 publications

References 37 publications

Assessment of In Vitro and In Silico Protocols for Sequence-Based Characterization of the Human Vaginal Microbiome

Assessment of In Vitro and In Silico Protocols for Sequence-Based Characterization of the Human Vaginal Microbiome

Impacts ofDesulfobacteralesandChromatialeson sulfate reduction in the subtropical mangrove ecosystem as revealed by SMDB analysis

MinION Sequencing of Yeast Mock Communities To Assess the Effect of Databases and ITS-LSU Markers on the Reliability of Metabarcoding Analysis

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