mag_recovery.md

Command examples for short-read MAG recovery

Assembly

• Shallow, short-read metagenomes were assembled with Megahit
• Using MFDxxxx as an example, the command would be:

megahit -t 10 -1 reads/MFDxxxx_R1.fastq.gz -2 reads/MFDxxxx_R2.fastq.gz -o contigs/MFDxxxx --k-list 27,43,71,99,127 -m 0.7 --min-contig-len 1000

Multi-sample coverage profiles

• To perform multi-sample metagenomic binning of paired-end short reads listed in MFD_forward.txt and MFD_reverse.txt, overlapping samples were determined using binchicken
• Sample overlaps are based on SingleM profiling in SingleM_outputs, about which more information is available in this GitHub repo
• Using MFDxxxx as the sample to be binned and MFDyyyy as an additional sample with an overlapping microbial community:

binchicken coassemble --forward-list MFD_forward.txt --reverse-list MFD_reverse.txt --sample-singlem-dir SingleM_outputs --max-coassembly-samples 1 --max-recovery-samples 10 --singlem-metapackage 2.1.GTDB_r214.metapackage_20231006.smpkg.zb

minimap2 -t 10 -ax sr contigs/MFDxxxx/final.contigs.fa reads/MFDyyyy_R1.fastq.gz reads/MFDyyyy_R2.fastq.gz | samtools view --threads 5 -Sb -F 2308 - | samtools sort --threads 3 - > map/MFDxxxx_MFDyyyy.bam
jgi_summarize_bam_contig_depths map/MFDxxxx_MFDyyyy.bam --percentIdentity 97 --outputDepth cov/MFDxxxx_MFDyyyy.tsv

• After calculating coverage values, the additional datasets can be appended:

cut -f4,5 cov/MFDxxxx_MFDyyyy.tsv > cov/MFDxxxx_MFDyyyy_trunc.tsv
paste -d "\t" cov/MFDxxxx_MFDxxxx.tsv cov/MFDxxxx_MFDyyyy_trunc.tsv > cov/MFDxxxx.tsv

Binning

• The contigs were binned into MAGs with MetaBat2
• Using MFDxxxx as an example, the command would be:

metabat2 -i contigs/MFDxxxx/final.contigs.fa -a cov/MFDxxxx.tsv -o bins/MFDxxxx/MFDxxxx -t 10 -m 1500 -s 100000 --saveCls