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The use of taxon-specific reference databases compromises metagenomic classification.

ABSTRACT: A recent article in BMC Genomics describes a new bioinformatics tool, HumanMycobiomeScan, to classify fungal taxa in metagenomic samples. This tool was used to characterize the gut mycobiome of hunter-gatherers and Western populations, resulting in the identification of a range of fungal species in the vast majority of samples. In the HumanMycobiomeScan pipeline, sequence reads are mapped against a reference database containing fungal genome sequences only. We argue that using reference databases comprised of a single taxonomic group leads to an unacceptably high number of false-positives due to: (i) mapping to conserved genetic regions in reference genomes, and (ii) sequence contamination in the assembled reference genomes. To demonstrate this, we replaced the HumanMycobiomeScan's fungal reference database with one containing genome sequences of amphibians and reptiles and re-analysed their case study. The classification pipeline recovered all species present in the reference database, revealing turtles (Geoemydidae), bull frogs (Pyxicephalidae) and snakes (Colubridae) as the most abundant herpetological taxa in the human gut. We also re-analysed their case study using a kingdom-agnostic pipeline. This revealed that while the gut of hunter-gatherers and Western subjects may be colonized by a range of microbial eukaryotes, only three fungal families were retrieved. These results highlight the pitfalls of using taxon-specific reference databases for metagenome classification, even when they are comprised of curated whole genome data. We propose that databases containing all domains of life provide the most suitable option for metagenomic species profiling, especially when targeting microbial eukaryotes.

SUBMITTER: R Marcelino V

PROVIDER: S-EPMC7045516 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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The use of taxon-specific reference databases compromises metagenomic classification.

R Marcelino Vanessa V Holmes Edward C EC Sorrell Tania C TC

BMC genomics 20200227 1

A recent article in BMC Genomics describes a new bioinformatics tool, HumanMycobiomeScan, to classify fungal taxa in metagenomic samples. This tool was used to characterize the gut mycobiome of hunter-gatherers and Western populations, resulting in the identification of a range of fungal species in the vast majority of samples. In the HumanMycobiomeScan pipeline, sequence reads are mapped against a reference database containing fungal genome sequences only. We argue that using reference database ...[more]

PMID: 32106809

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Project description:Many environmental and biomedical biomonitoring and detection studies aim to explore the presence of specific organisms or gene functionalities in microbiome samples. In such cases, when the study hypotheses can be answered with the exploration of a small number of genes, a targeted PCR-approach is appropriate. However, due to the complexity of environmental microbial communities, the design of specific primers is challenging and can lead to non-specific results. We designed PhyloPrimer, the first user-friendly platform to semi-automate the design of taxon-specific oligos (i.e., PCR primers) for a gene of interest. The main strength of PhyloPrimer is the ability to retrieve and align GenBank gene sequences matching the user's input, and to explore their relationships through an online dynamic tree. PhyloPrimer then designs oligos specific to the gene sequences selected from the tree and uses the tree non-selected sequences to look for and maximize oligo differences between targeted and non-targeted sequences, therefore increasing oligo taxon-specificity (positive/negative consensus approach). Designed oligos are then checked for the presence of secondary structure with the nearest-neighbor (NN) calculation and the presence of off-target matches with in silico PCR tests, also processing oligos with degenerate bases. Whilst the main function of PhyloPrimer is the design of taxon-specific oligos (down to the species level), the software can also be used for designing oligos to target a gene without any taxonomic specificity, for designing oligos from preselected sequences and for checking predesigned oligos. We validated the pipeline on four commercially available microbial mock communities using PhyloPrimer to design genus- and species-specific primers for the detection of Streptococcus species in the mock communities. The software performed well on these mock microbial communities and can be found at https://www.cerealsdb.uk.net/cerealgenomics/phyloprimer.

Dataset Information

The use of taxon-specific reference databases compromises metagenomic classification.

Publications

The use of taxon-specific reference databases compromises metagenomic classification.

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