Bio Applied Assembly Binning

Workflow Overview

reads → MEGAHIT assembly → filter ≥500 bp contigs
      → map reads back → jgi_summarize_bam_contig_depths
      → MetaBAT2/CONCOCT/MaxBin2 binning
      → DAS_Tool dereplication
      → CheckM quality assessment
      → filter HQ MAGs (≥90% complete, <5% contamination)

MEGAHIT Assembly

megahit \
    -1 decontam_1.fastq.gz \
    -2 decontam_2.fastq.gz \
    -o megahit_assembly/ \
    --min-contig-len 500 \
    -t 16 \
    -m 0.5              # use at most 50% of available RAM

# Check stats
seqkit stats megahit_assembly/final.contigs.fa

Key parameter: --k-list 21,29,39,59,79,99,119,141 (default). Multi-k handles extreme coverage variation (1x rare organism vs 1000x abundant organism in the same community).

Coverage Estimation

# Index assembly and map reads back
bowtie2-build megahit_assembly/final.contigs.fa contigs_index

bowtie2 -x contigs_index -1 decontam_1.fastq.gz -2 decontam_2.fastq.gz \
    -p 16 --no-unal | samtools sort -@ 8 -o contigs_mapped.bam
samtools index contigs_mapped.bam

# Generate depth file (required by MetaBAT2)
jgi_summarize_bam_contig_depths \
    --outputDepth depths.txt \
    contigs_mapped.bam
# Output columns: contigName, contigLen, totalAvgDepth, sampleDepth, sampleDepthVar

Contig Binning

# MetaBAT2
metabat2 \
    -i megahit_assembly/final.contigs.fa \
    -a depths.txt \
    -o bins/bin \
    --minContig 1500 \
    --minClsSize 100000 \   # minimum 100 kb bin size
    -t 8 \
    --saveCls

# Run all three binners then combine with DAS_Tool
das_tool \
    -i metabat2_bins,concoct_bins,maxbin2_bins \
    -l MetaBAT2,CONCOCT,MaxBin2 \
    -c megahit_assembly/final.contigs.fa \
    -o dastool_output/ \
    -t 8 \
    --write_bins

Binning signals used by MetaBAT2:

1. Tetranucleotide frequency (TNF) — 256-dimensional composition vector, genome-specific
2. Coverage depth across samples — organism-specific abundance

MAG Quality Assessment (CheckM)

checkm lineage_wf bins/ checkm_out/ -x fa -t 16 --pplacer_threads 4
checkm qa checkm_out/lineage.ms checkm_out/ -o 2 > checkm_summary.txt

MIMAG Quality Tiers (Bowers et al. 2017, Nature Biotechnology)

CheckM uses ~100 conserved single-copy marker genes expected exactly once in any complete bacterial genome. Marker gene set is selected based on phylogenetic placement of each bin.

Binner Comparison