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QC Statistics

Using CheckM2 on long prokaryotic contigs

The easiest way to filter out long (>300 kb) erroneous prokaryotic contigs is to run CheckM2 on individual contigs.

If a chimeric contig is long enough, it will often have high CheckM contamination due to the presence of duplicated marker genes from multiple organisms.

To extract all contigs of length >= X bp and run CheckM2, you can do (with mylotools)

cd myloasm_results
mylotools extract-contigs --output-folder contigs_dir --min-contig-length X
checkm2 predict --input contigs_dir -x fa -o checkm2_results --threads 40

Using k-mer multiplicity statistics for duplicates/strain chimeras

Myloasm's fasta outputs have information about how often 21-mers are repeated (its multiplicity) within a contig:

>u123123ctg_XXX_mult-1.00 <- fasta record with k-mer multiplicity

In our experience, prokaryotic contigs should almost always have average k-mer multiplicity near 1.00. If you have a very long contig (> 1M bp) of multiplicity > 1.05, it may be a chimera from multiple strains of a species.

For small genomes (e.g. viruses), the expected k-mer multiplicity may deviate from 1.00. However, a small contig with k-mer multiplicity >> 1 can be suspicious. A contig with k-mer multiplicity = 2, 3, or an integer multiple can indicate a perfectly duplicated contig.

Notes on circularized contigs

For prokaryotic genomes, low CheckM2 completeness can indicate premature circularization. However:

  • we have found that complete organelle genomes (mitochondria, plastids from microeukaryotes) can have non-trivial CheckM2 completeness (> 30% but < 90%)
  • secondary chromosomes and genomes with multiple chromosomes can have lower completeness
  • some clades of microbes have low CheckM2 completeness scores, even when they're complete

The myloasm tag circular-possibly indicates lower confidence circular genomes (due to low coverage or assembly graph ambiguity), but these can often be complete genomes, especially if CheckM2 scores are good.