Abstract

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.

Full Text
Published version (Free)

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call