Entering edit mode
4.9 years ago
hafiz.talhamalik
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350
I have fastq files of microbial genomics data. and want to get plasmids from it. I have tried plasmidfinder and spadesplasmid as well. but all the time file show up empty. why is it so ? any suggestion ?
How was the DNA prepared?
Do you have a reference sequence for any plasmids you can blast contigs against?
Do you know for certain this organism, under the conditions you tested, contains a plasmid?
The DNA was extracted by using whole DNA extraction kit under optimal conditions. Yes, We do have reference sequence of a plasmid.
I think its still possible to lose plasmids (especially if they’re small), when doing preps which purport to be “total DNA preps” (but I’m not convinced this is definitively the answer anyway).
It may be worth taking a step back, and just doing a ‘normal’ de novo assembly, with no special consideration for the plasmids, and then BLAST all the contigs against the reference plasmids sequence. Step one is figuring out whether you even have the corresponding DNA there at all, before plasmidSPAdes etc, to see if they have anything to work with.
I tried aligning fastq file with plasmid sequence and found some hits as well but when I annotated those hits with plasidfinder I found nothing there.
I would assemble before you BLAST - so BLAST contigs instead of reads.
Plasmid assemblers aren't magic however, so assuming you're doing nothing wrong, there's still no guarantee they'll find what you're looking for in a neat way.
What exactly have you tried? Can you show the commands?
I tried assembly using velvet, spades and unicycler. then I used assembled contigs for plasmid finding for web based plasmidfiner.
for plasmid spades I used this command.