mfd_novelty_section

This repo is part of the Microflora Danica (MFD) project and it reproduces the results from "Section VI: MAGs from Danish habitats double the known species fraction in metagenomes" of the MFD manuscript.

Input data

Please download the input file from Zenodo.

The SingleM metapackakge with GTDB R214 supplemented with other genomes can be downloaded from this Zenodo.

The SingleM metapackakge with GTDB R214 supplemented with other genomes AND our short-read (SR) MFD MAGs can be downloaded from this Zenodo.

Codes and scripts

All SingleM condensed profiles from our big batch samples are merged together using the script: /scripts/00-merge_condensed_singlem_outputs.r.

R scripts used for generating SingleM-related plots (eg., Figure 5a, Figure 5b, Supplementary Figure 6, and Supplementary Figure 7) in the paper are in the folder /scripts.

Codes for generating Figure 5c can be found in this repo.

SingleM example commands:

Taking "sample1" and metapackage "uhgg_smag_spire_oceans_gems.smpkg" as an example:

1. SingleM microbial profiling: SingleM pipe metagenome

singlem pipe
-1 /PATH_TO_FORWARD_READ/sample1_trimmed.fastq.gz
-2 /PATH_TO_REVERSE_READ/sample1_trimmed.fastq.gz
--threads 10
--metapackage /PATH_TO_FILE/uhgg_smag_spire_oceans_gems.smpkg
--otu-table sample1.otu_table.csv
--archive-otu-table sample1.otu_table.csv.archive
--taxonomic-profile sample1.condensed_otu_defaultcutoffs

2. SingleM microbial fraction approximation

singlem microbial_fraction
--input-profile sample1.condensed_otu_defaultcutoffs
--output-tsv sample1_readfrac_stdout.tsv
--output-per-taxon-read-fractions sample1_readfrac_pertaxon
-1 /PATH_TO_FORWARD_READ/sample1_trimmed.fastq.gz
-2 /PATH_TO_REVERSE_READ/sample1_trimmed.fastq.gz
--metapackage /PATH_TO_FILE/uhgg_smag_spire_oceans_gems.smpkg

3. SingleM for MAG recovery of microbial community approximation

Taking "MAG1" as an example:

3a. SingleM pipe MAGs

singlem pipe
--genome-fasta-files /PATH_TO_FILE/MAG1.fa
--otu-table MAG1_genome.otu_table.csv
--threads 2
--archive-otu-table MAG1_genome.otu_table_archive.csv
--taxonomic-profile MAG1_condensed_otu_defaultcutoffs
--metapackage /PATH_TO_FILE/uhgg_smag_spire_oceans_gems.smpkg

3b. SingleM summarise to combine otu tables of all MAGs

singlem summarise
--input-otu-tables-list /PATH_TO_FILE/ABSOLUTE_FILEPATHS_TO_MAG_OTUTABLES_LIST.txt
--output-otu-table genomes.otu_table_combined.csv
--metapackage /PATH_TO_FILE/uhgg_smag_spire_oceans_gems.smpkg

3b. SingleM summarise to combine otu tables of all metagenomes

singlem summarise
--input-otu-tables-list /PATH_TO_FILE/ABSOLUTE_FILEPATHS_TO_METAGENOME_OTUTABLES_LIST.txt
--output-otu-table metagenome_otu_table_combinedotu.txt
--metapackage /PATH_TO_FILE/uhgg_smag_spire_oceans_gems.smpkg

3c. SingleM appraise

singlem appraise
--metagenome-otu-tables /PATH_TO_FILE/metagenome_otu_table_combinedotu.txt
--genome-otu-tables /PATH_TO_FILE/genomes.otu_table_combined.csv
--metapackage /PATH_TO_FILE/uhgg_smag_spire_oceans_gems.smpkg
--output-binned-otu-table appraise_binned_otu_table.csv
--output-unbinned-otu-table appraise_unbinned_otu_table.csv
--output-unaccounted-for-otu-table appraise_unaccounted_for.csv
--output-found-in &> singlem_appraise.log &

Additional note:

SingleM results on NCBI datasets come from:

SingleM and Sandpiper: Robust microbial taxonomic profiles from metagenomic data., Ben J. Woodcroft, Samuel T. N. Aroney, Rossen Zhao, Mitchell Cunningham, Joshua A. M. Mitchell, Linda Blackall, Gene W. Tyson., bioRxiv 2024.01.30.578060; doi: https://doi.org/10.1101/2024.01.30.578060

The microbial_fraction module of SingleM:

Eisenhofer, Raphael, Antton Alberdi, and Ben J. Woodcroft. Large-scale estimation of bacterial and archaeal DNA prevalence in metagenomes reveals biome-specific patterns. bioRxiv (2024): 2024-05. https://doi.org/10.1101/2024.05.16.594470

Citation of MFD manuscript:

Microflora Danica: the atlas of Danish environmental microbiomes., CM Singleton, TBN Jensen, F Delogu, EA Sørensen, VR Jørgensen, SM Karst, Y Yang, KS Knudsen, M Sereika, F Petriglieri, S Knutsson, SM Dall, RH Kirkegaard, JM Kristensen, BJ Woodcroft, DR Speth, STN Aroney, The Microflora Danica Consortium, M Wagner, MKD Dueholm, PH Nielsen, M Albertsen., bioRxiv 2024.06.27.600767; doi: https://doi.org/10.1101/2024.06.27.600767