Hi,
https://github.com/phac-nml/mob-suite
I have a plasmid - it is 45,000bp IncX3 type I tried reconstructing it from a sample where I know it is present, using mob_suite recon using default parameters.
The results indicate that there is a MOBP gene, IncX3 rep, size 40,000. Yet, it does not consider it a plasmid:
molecule_type size rep_type(s) rep_type_accession(s) relaxase_type(s) relaxase_type_accession(s) mpf_type mpf_type_accession(s)
chromosome 36253 IncX3 000221__NC_025130_00012 MOBP NC_020552_00059 MPF_T,MPF_T,MPF_T,MPF_T,MPF_T NC_023277_00163,NC_019157_00029,NC_019157_00030,NC_019083_00030,NC_017624_00009
What can I change in the parameters to get the plasmid?
I tried using a min_lnegh 500 and it didn't help - is it a DB problem?
Thanks,
1 answer
The thing worth noticing is that your markers were found but the molecule call wasn't. Those are two different steps in mob_recon: replicon, relaxase and mpf come from separate marker searches, while molecule_type comes from whether the contig gets assigned to a plasmid cluster by mash against the bundled reference database. Markers present plus a chromosome call means the clustering step failed, not the marker step, so min_length was never going to help you there.
Have a look at primary_cluster_id and mash_nearest_neighbor / mash_neighbor_distance in that same contig_report. If the cluster id is empty and the mash distance sits above threshold, then either nothing in the bundled DB was close enough (which is your database question, and it's a real possibility for a divergent plasmid) or the contig isn't purely plasmid.
Which brings up the other thing. You say the plasmid is 45 kb but the molecule is 36,253. That 9 kb gap seems worth chasing before touching any parameters. Either the remainder is sitting on other contigs, or the contig is a plasmid-chromosome chimera, which short-read assemblers do quite happily through shared IS elements, and IncX3 plasmids are IS-rich. If it is chimeric then the chromosome call is arguably correct rather than a bug. Two cheap checks: compare that contig's read depth against your chromosomal contigs, because a 45 kb plasmid sitting at the same coverage as the chromosome is suspicious, and blast the contig against a known IncX3 reference to see where the alignment stops.
If the assembly came out of Unicycler there's a --unicycler_contigs option that lets mob_recon use the circularity information in the headers, which fixes a fair number of cases like this. Long reads would settle it outright if you have any.
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When referring to programs that are not commonly used it may be best to provide a link to the said program. Looks like you are referring to: https://github.com/phac-nml/mob-suite
Did you look at the output report files (
contig_report.txtbased on the description at GitHub site)? For some reason the program is identifying your molecule as chromosome and perhaps there is a clue in the output files as to why.The information I added (the table) is from the contig_report.txt it shows the IncX3 plasmid rep and MOBP
I added the github link above