How to determine the sequence of the plasmids from ngs results?

Question

[silvia.caprari84]

Hi all,

I am trying to interpret the results coming from ngs on some bacterial isolates that are antibiotic resistant. This is completely new for me and I have different questions about it.I would like to know; how I can reconstruct the sequence of the plasmids starting from the contigs I have? I mean, I have different contigs..should each contig correspond to a different plasmid?and if not, how can I "re-build" the entire sequence of the plasmid from these contigs?

Then..In order to see if particular genes were present in my isolates, I run Blast by using the sequence of a known gene as a query against the FASTA file containing all the contigs..As a result, I have different contigs that match with the known sequence..does it mean that I have the plasmid sequence split in different contigs??or anything else?

furthermore, i find the same sequence alignment with different contigs..so it seems like the same sequence is repeated in different contigs(??)what does it mean??that more contigs containing the same sequence can be generated by the sequencing procedure?

Thank you very much

[Benn]

Did you try to align the contigs that match the same blast hit? With clustalW for example? You can see what the difference (and similarity) is between these contigs in a multiple alignment.

[sidrairshad29]

silvia, have you assembled a complete genome from your contigs? I am working on same stuff. I have my sequence in contigs form and i want a complete genome of it. Let me know if you know the answer.

Answer 1

[silvia.caprari84]

Ok. I will try.Can I ask why I find contigs that match the same known sequence (when I run Blast) but they show slightly differences in terms of their nucleotide sequence? For example I have two contigs that match the known sequence for the same region, one matches it with 100% identity , the other one with 99%...Does it mean that normally I have more possible contigs for the same sequence?? on what does it depend? The sequencing was carried out with Illumina.

[Benn]

It's hard to say, why.

The differences you find between your contigs might be caused by many factors, e.g.,

• mutations
• PCR errors
• sequencing errors
• assembly settings

And maybe even more...

Answer 2

[silvia.caprari84]

so, I don't know if I am talkin about fantasy..but could it have been the result of more copies of the same plasmid in the cell? some of these copies underwent to mutations and some other didn't?

[Benn]

That might be, but is hard to tell from the information you provided. It might also be that you have a culture and a fraction of your population is mutated and others not. Or you might be looking at sequencing errors...

[Benn]

If you really think you have two different plasmids, and they are interesting to look into further. Try to isolate these plasmids and sequence them the old fashion way (Sanger).