Bowtie paired-end mapping: insert size issue
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4 months ago
biostart ▴ 370

Hi all,

I have 150-bp paired-end sequencing reads, and I am mapping them with Bowtie with the following parameters:

 bowtie -v 2 -p 16 -m 1 --minins 0 --maxins 1000 --best --allow-contain --solexa-quals


However, I get as output only fragment sizes >150bp. My thinking was that parameter --minins 0 tells Bowtie to accept all small DNA fragments, but apparently it's not?

Could you please advise how to adjust Bowtie parameters to get all the smaller fragments in this mapping?

Thanks!

alignment mapping bowtie • 506 views
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Is this data trimmed? If you expect small inserts then your R1/R2 reads are going to overlap. Perhaps bowtie is having trouble with those inserts. Are you using an old dataset? solexa-quals should not be applicable to datasets of recent vintage.

BTW - default value for --minins is already 0.

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Thank you, I will remove "solexa-quals". The data is not trimmed. I am expecting a significant portion of reads smaller than 150bp (which is smaller than the insert size). Do you think Bowtie just can not map them unless they are trimmed down to smaller sizes? It would be pity to remove a huge part of the reads this way.

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If you expect a lot of your reads to overlap then you may want to use program like bbmerge to merge the reads and then trim merged read to remove adapter sequences. You will end up with one read after the merging/trimming. bowtie (v.1.x) does ungapped alignments and will have trouble aligning if you have adapter sequences in your reads.

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Adapters are already removed by the sequencing facility, what I have are pure 150-bp sequenced reads without adapters. If you think trimming them is the only option, I can try to decrease their sizes from 150 to 50 (still pity to lose such amount of data...)

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If that is the case I don't know what you mean by

I am expecting a significant portion of reads smaller than 150bp

What kind of data is this? bowtie v.1.x is generally used for smallRNA data (where you want ungapped alignments). If that is not your data then you should use a different aligner. Also check a few reads that are not aligning to make sure they are from correct genome by blasting then at NCBI.

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sorry, I meant to say I am expecting a significant portion of DNA fragments shorter than the 150bp sequencing length. This is MNase-seq data, but our pipeline depends on Bowtie for historic reasons and it would be difficult to change it to another aligner.

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I see. So you have analyzed this kind of data before and this time something looks different?

How are you determining following? Based on alignments?

I get as output only fragment sizes >150bp

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Yes, before we did 100bp paired-end sequencing and the mapping was OK with those parameters. Reads <100bp were anyway usually outside of our interest. But now the new sequencing was done with 150bp paired-end sequencing and it started interfering with the analysis. Now the fragment sizes are only >150bp, I see this in the Bowtie output after the alignment is done.

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