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helelein 07-17-2014 10:01 AM

How to blast a transposon sequence to a MiSeq transposon library
Hi there!
I'm brand new on the forum and in massive parallel sequencing analysis too!

I have sequenced 2 transposon libraries from a bacterial genome (input/output pool) with MiSeq from Illumina. My interest is to find where the transposon has been inserted in the bacterial genome. For the sequencing, DNA was broken into small pieces and transposon containing fragments were captured with a biotinilated probe.

Before blasting my transposon sequences to the bacterial genome, I need to check my sequences and get rid of the ones that don't have it. Therefore I would like to make a blast of my sequences or align them with the end of the sequence of the transposon; in order to get rid of the sequences not containing the transposon.

Could anyone give me a hint how to perform such a blast, please? I'm trying with CLC workbench but don't manage to do it. Since usually a blast is done against a database and I just want to blast a single sequence.


Brian Bushnell 07-17-2014 11:33 AM

Blast may not be the best tool for this. I suggest BBDuk, which is designed rapidly filter millions of reads based on kmer-matches.

In linux/bash, the command would be: -Xmx1g in=reads.fq out=unmatched.fq outm=matched.fq ref=transposon.fa k=31

Reads with 31-mers matching the transposon sequence will go to "matched.fq"

If you have paired reads in 2 files, use the "in2" and "outm2" flags.

helelein 07-17-2014 12:15 PM

Thanks Brian for your suggestion!
I'm trying to run your bbduk, but don't manage to use it. I'll ask for help tomorrow morning to an informatician in my group.

Brian Bushnell 07-17-2014 04:06 PM

OK. If you need help from me, please post the error message (if any) and the operating system you are using.

helelein 07-22-2014 07:38 AM

Thanks for your help Brian, it worked perfectly.

I have another question. I would like to get rid of the transposon fragment in the sequences that got it, in order to blast them afterwards with my bacterial genome. Is there another tool in your bbmap software that could help me?

Brian Bushnell 07-22-2014 09:52 AM

Blast is a local aligner, so removing transposon fragments should not be necessary unless there is more transposon than read and the transposon sequence is in your blast database, which it shouldn't be

Unfortunately, I don't have a good tool for that right now. BBDuk can remove sequences from the ends of reads, but you have to know which end the sequence is on. I will modify it so that it can remove from either end more easily. But for now, I suggest you just go ahead and blast and not worry about it unless you see a problem.

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