How do we know that our contig sequence is really plasmid?

Question

[rickyalfaray]

Hi everyone,

I was confused about the method to confirm whether our contig sequence is really a plasmid or not. I have downloaded the whole genome sequence assembly (either from long read or short read) from a bacterial species, which was H. pylori. This bacteria is known to have a very low copy number of plasmid and somehow the plasmid itself is not easy to detect especially based on the whole genome sequence. The method I used was just blasted by using Abricate software against the plasmid databases (such as PLSDB, plasmidfinder, and I made my own database by downloading all plasmid sequences from NCBI/other public databases). I settled the coverage and identity by 90.

After I had detected some plasmids, I tried to extract the plasmids contigs from the WGS data. I got several types of plasmids, but one type of these plasmids seems to have GC content around 38-39 which is a little bit high for plasmids.

My questions are:

1. How do we know that our contig sequence (that we extracted from the assembled WGS) is really plasmid?
2. How do we check whether these contig sequences are extrachromosomal bacteria?
3. Is there any tool to do predict whether our contig sequence is a plasmid or not?

Thank you

Kind regards,
Ricky

[cpad0112]

i think most of them are sequence based detection which you already have done. May be you can trying using multiple plasmid identification tools (plasmidseeker, plasmidspades etc) and take a consensus of the findings or use a complementary experimental technique to confirm your findings.

[rickyalfaray]

Thank you. I already did use plasmidseeker as your suggestion. I also tried to validate manually one by one. Now it becomes clear. Thank you.

Answer 1

[Mensur Dlakic]

In addition to sequence comparison methods - which should always be tried - there are approaches based on statistical properties of plasmid vs. chromosomal sequences.

• https://github.com/zhenchengfang/PPR-Meta
• https://github.com/smaegol/PlasFlow
• https://github.com/bioinfo-ut/PlasmidSeeker
• https://github.com/aldertzomer/RFPlasmid

[rickyalfaray]

Thank you. I already performed the detection using PlasmidSeeker as your suggestion, also Plasmid finder and Platon, and I got some new plasmids. I tried to confirm manually by seeing their genes GC content, annotation, etc. I exclude the ambiguous results. Now I confident that they are plasmids. For validation, I also tried to extract some plasmids that were not yet reported in the databases but detected by the software, and I could get the plasmids. Thank you for your suggestion.

Answer 2

[oschwengers]

You could search for plasmid-specigic motifs like Inc types (incompatibility) and origins of transfer (oriT).

Wonderful tools to use are PlasmidFinder and MOB-suite.

In addition to the tools mentioned in the other answer:

• https://github.com/wandreopoulos/deeplasmid
• https://github.com/oschwengers/platon
• https://github.com/labgem/PlaScope

[rickyalfaray]

Thank you. I already tried Plasmid finder and Platon as your suggestion, also PlasmidSeeker, and I found new plasmids. I tried to confirm manually each plasmid that I found also. Some plasmids were also I confirmed by experimental study. Now I am confident. Thank you.