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Sensitivity of shotgun metagenomics to host DNA: abundance estimates depend on bioinformatic tools and contamination is the main issue

A recent study reported that increasing host DNA abundance and reducing read depth impairs the sensitivity of detection of low-abundance micro-organisms by shotgun metagenomics. The authors used DNA from a synthetic bacterial community with abundances varying across several orders of magnitude and a...

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Autores principales: McArdle, Andrew J., Kaforou, Myrsini
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Microbiology Society 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7523627/
https://www.ncbi.nlm.nih.gov/pubmed/33005868
http://dx.doi.org/10.1099/acmi.0.000104
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author McArdle, Andrew J.
Kaforou, Myrsini
author_facet McArdle, Andrew J.
Kaforou, Myrsini
author_sort McArdle, Andrew J.
collection PubMed
description A recent study reported that increasing host DNA abundance and reducing read depth impairs the sensitivity of detection of low-abundance micro-organisms by shotgun metagenomics. The authors used DNA from a synthetic bacterial community with abundances varying across several orders of magnitude and added varying proportions of host DNA. However, the use of a marker-gene-based abundance estimation tool (MetaPhlAn2) requires considerable depth to detect marker genes from low-abundance organisms. Here, we reanalyse the deposited data, and place the study in the broader context of low microbial biomass metagenomics. We opted for a fast and sensitive read binning tool (Kraken 2) with abundance estimates from Bracken. With this approach all organisms are detected even when the sample comprises 99 % host DNA and similarly accurate abundance estimates are provided (mean squared error 0.45 vs. 0.3 in the original study). We show that off-target genera, whether contaminants or misidentified reads, come to represent over 10 % of reads when the sample is 99 % host DNA and exceed counts of many target genera. Therefore, we applied Decontam, a contaminant detection tool, which was able to remove 61 % of off-target species and 79 % of off-target reads. We conclude that read binning tools can remain sensitive to low-abundance organisms even with high host DNA content, but even low levels of contamination pose a significant problem due to low microbial biomass. Analytical mitigations are available, such as Decontam, although steps to reduce contamination are critical.
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spelling pubmed-75236272020-09-30 Sensitivity of shotgun metagenomics to host DNA: abundance estimates depend on bioinformatic tools and contamination is the main issue McArdle, Andrew J. Kaforou, Myrsini Access Microbiol Short Communication A recent study reported that increasing host DNA abundance and reducing read depth impairs the sensitivity of detection of low-abundance micro-organisms by shotgun metagenomics. The authors used DNA from a synthetic bacterial community with abundances varying across several orders of magnitude and added varying proportions of host DNA. However, the use of a marker-gene-based abundance estimation tool (MetaPhlAn2) requires considerable depth to detect marker genes from low-abundance organisms. Here, we reanalyse the deposited data, and place the study in the broader context of low microbial biomass metagenomics. We opted for a fast and sensitive read binning tool (Kraken 2) with abundance estimates from Bracken. With this approach all organisms are detected even when the sample comprises 99 % host DNA and similarly accurate abundance estimates are provided (mean squared error 0.45 vs. 0.3 in the original study). We show that off-target genera, whether contaminants or misidentified reads, come to represent over 10 % of reads when the sample is 99 % host DNA and exceed counts of many target genera. Therefore, we applied Decontam, a contaminant detection tool, which was able to remove 61 % of off-target species and 79 % of off-target reads. We conclude that read binning tools can remain sensitive to low-abundance organisms even with high host DNA content, but even low levels of contamination pose a significant problem due to low microbial biomass. Analytical mitigations are available, such as Decontam, although steps to reduce contamination are critical. Microbiology Society 2020-02-17 /pmc/articles/PMC7523627/ /pubmed/33005868 http://dx.doi.org/10.1099/acmi.0.000104 Text en © 2020 The Authors http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons.
spellingShingle Short Communication
McArdle, Andrew J.
Kaforou, Myrsini
Sensitivity of shotgun metagenomics to host DNA: abundance estimates depend on bioinformatic tools and contamination is the main issue
title Sensitivity of shotgun metagenomics to host DNA: abundance estimates depend on bioinformatic tools and contamination is the main issue
title_full Sensitivity of shotgun metagenomics to host DNA: abundance estimates depend on bioinformatic tools and contamination is the main issue
title_fullStr Sensitivity of shotgun metagenomics to host DNA: abundance estimates depend on bioinformatic tools and contamination is the main issue
title_full_unstemmed Sensitivity of shotgun metagenomics to host DNA: abundance estimates depend on bioinformatic tools and contamination is the main issue
title_short Sensitivity of shotgun metagenomics to host DNA: abundance estimates depend on bioinformatic tools and contamination is the main issue
title_sort sensitivity of shotgun metagenomics to host dna: abundance estimates depend on bioinformatic tools and contamination is the main issue
topic Short Communication
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7523627/
https://www.ncbi.nlm.nih.gov/pubmed/33005868
http://dx.doi.org/10.1099/acmi.0.000104
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