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R1 and R2 matching

Dear all,

I have a number of X_R1_001.fastq.gz X_R2_001.fastq.gz and I would to match them. I have seen a pipeline where they give this: 

# R1 and R2 matching with Compare.pl: for i in Filter-Cutadapt-Demul*.fastq; do perl Compare.pl -f $i -r Filter-Cutadapt-R2.fastq -of Paired-$i -or R2-Paired-$i -os Single-$i; done

but the perl script is not given. Does anybody know how to match the fastq.gz files? I would like trying matching them because when I run the bwa mem I get this: 

[M::process] read 66226 sequences (10000126 bp)...[M::process] read 66226 sequences (10000126 bp)..[M::mem_pestat] # candidate unique pairs for (FF, FR, RF, RR): (0, 144, 0, 0)[M::mem_pestat] skip orientation FF as there are not enough pairs[M::mem_pestat] analyzing insert size distribution for orientation FR...[M::mem_pestat] (25, 50, 75) percentile: (225, 294, 355)

I am not sure the solution is matching the fastq files, does anybody have a better solution? thanks in advance

next-gen bwa
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updated by GenoMax GenoMax • written by ieie • ieie

What makes you believe that your two files are not matched? Normally R1 and R2 files are already matched, with which I mean the order of the reads are the same in both files.

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written by Benn Benn

thanks! I thought since in BWA it was not finding the pairs there was a problem with the matching.

• 1 views • link
written by ieie • ieie

bwa seems to find F-R (forward reverse) orientated reads, which is the most logical orientation for paired end reads.

• 1 views • link
written by Benn Benn

Hi There,

Can you please explain what you mean by matching reads?

• 1 views • link
written by Sej Modha Sej Modha

matching the reads in a way that bwa mem can find the pairs when it runs. Another thing that I don't understand is that after running:

bwa -M -B 4 reference.fas x_R1.fastq.gz  x_R2.fastq.gz > x.sam

and then I run bwa mem only for the R1 file:

bwa -M -B 4 reference.fas x_R1.fastq.gz > x.sam

when I look at both bam files I see a max coverage of 3000 for the R1 alignment and 6000 for the pair ended alignment, is it correct? because since bwa is matching I was expecting the same coverage, 3000.

• 1 views • link
written by ieie • ieie

Hello ieie ,

it's not clear to me what's your problem. The message you see by bwa mem is just a normal information. It's neither an error nor a warning.

fin swimmer

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written by finswimmer finswimmer

My problem is that I was thinking there was an error in bwa while trying to find the pairs. But I understand now that this messages are just saying that it skips the FF or FR orientation because there are not enough pairs but uses another orientation. thanks for your answer.

• 1 views • link
written by ieie • ieie

1 answer

Use repair.sh from BBMap suite. It will allow you to properly pair R1/R2 reads in the files and collect signleton reads in a separate file.

You should never trim your R1/R2 reads independently, if that is what led to this problem.

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written by GenoMax GenoMax

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