Extending and improving metagenomic taxonomic profiling with uncharacterized species with MetaPhlAn 4

Abstract

Metagenomic assembly enables novel organism discovery from microbial communities, but from most metagenomes it can only capture few abundant organisms. Here, we present a method - MetaPhlAn 4 - to integrate information from both metagenome assemblies and microbial isolate genomes for improved and more comprehensive metagenomic taxonomic profiling. From a curated collection of 1.01M prokaryotic reference and metagenome-assembled genomes, we defined unique marker genes for 26,970 species-level genome bins, 4,992 of them taxonomically unidentified at the species level. MetaPhlAn 4 explains ~20% more reads in most international human gut microbiomes and >40% in less-characterized environments such as the rumen microbiome, and proved more accurate than available alternatives on synthetic evaluations while also reliably quantifying organisms with no cultured isolates. Application of the method to >24,500 metagenomes highlighted previously undetected species to be strong biomarkers for host conditions and lifestyles in human and mice microbiomes, and showed that even previously uncharacterized species can be genetically profiled at the resolution of single microbial strains. MetaPhlAn 4 thus integrates the novelty of metagenomic assemblies with the sensitivity and fidelity of reference-based analyses, providing efficient metagenomic profiling of uncharacterized species and enabling deeper and more comprehensive microbiome biomarker detection.

Variability of strain engraftment and predictability of microbiome composition after fecal microbiota transplantation across different diseases

“…We implemented the approach and the species-specific cut-offs that define strain identity within StrainPhlAn 4 (ref. 39 ), which we empowered with a custom database of marker gene sequences from around 729,000 microbial genomes and metagenome-assembled genomes (MAGs). With such references, StrainPhlAn is able to detect and model strains belonging to a total of 4,992 yet-to-be-characterized species 25 ; that is, unknown SGBs (uSGBs; Methods).…”

“…Species-level profiling was performed on samples sequenced to a depth higher than 1 Gbp (n = 1,419; 100 samples being excluded from downstream analyses) using MetaPhlAn 4 (ref. 23,39 ) with default parameters and the custom extended SGB database. uSGBs with fewer than five MAGs were discarded, as there is a higher risk of them being the result of assembly artifacts or chimeric sequences.…”

“…Strain profiling was performed with a modified version of StrainPhlAn 3 (ref. 23 ) using the custom SGB marker database described above that has been released as StrainPhlAn 4 39 . We modified the StrainPhlAn code to change the sample and marker filtering behavior to allow for profiling more samples and SGBs.…”

“…We profiled 9,900 healthy human microbiome samples from 59 datasets spanning different body sites (airways, gastrointestinal tract, oral, skin and urogenital tract; Supplementary Table 11) using MetaPhlAn 4 (ref. 23,39 ) with default parameters and the custom SGB database (see above). Only individuals older than 3 years and from cohorts involving industrialized nonrural populations (defined as 'Westernized' in cMD 78 ) were considered.…”

Meta-Omics Analysis of a Formate Sidestream-Mediated Partial Nitritation-Coupled Anammox Process

“…The abundance and expression of functional genes were determined using DIAMOND (version 0.9.13) by comparing sample DNA and RNA reads against a custom, manually curated database, which containing 13 nitrogen cycle key enzymes and 3 formate anabolic/catabolic enzymes. Metaphlan (version 3.0.7) with a microbial database (version mpa_v30_CHOCOPhlAn_201901) was used for the taxonomic assignment analysis based on DNA reads.…”