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output of bedtools bamtofastq are empty files

Hi All,

I would like to convert my bam file to fastq. But after I run bedtools with the sorted bam file, I've got two empty files. What should be wrong? (Or what is the proper way to convert a mapped paired-end bam file to fastq?)

What I tried:

samtools sort -n /pathtothefiles/NA12878.chr1.bam /pathtothefiles/NA12878_chr1.qsort
bedtools bamtofastq -i /pathtothefiles/NA12878_chr1.qsort \
                      -fq aln.end1.fq \
                      -fq2 aln.end2.fq

Thanks in advance!

bamtofastq bam fastq samtools bedtools
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updated by Ram Ram • written by anon • anon

Samtools doesn't sort files in place, it writes them sorted to a new file. You need something like samtools sort -n foo.bam > name_sorted.bam

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written by Devon Ryan Devon Ryan

sorry, I deleted too much from the inserted command ... I did it properly, like you wrote.

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written by anon • anon

OK, thanks for the update. I've moved my answer to a comment in hopes that someone more familiar with that particular command will answer.

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written by Devon Ryan Devon Ryan

it's fairly possible that you can skip the qsort step, read the file from samtools and execute bedtools next. Also, it's possible that samtools is still running and the files will be available at the end of the run. Do you see the file's size increasing with time? Please edit your post with Code paragraph so we can read more easily.

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updated by Ram Ram • written by madkitty madkitty

I usually use bam2fq instead of bedtools, did you try the following?

htscmd bamshuf -uOn 128 aln_reads.bam tmp > shuffled_reads.bam
htscmd bam2fq -a shuffled_reads.bam > interleaved_reads.fq
gzip interleaved_reads.fq

In one line: 

tscmd bamshuf -uOn 128 aln_reads.bam tmp | htscmd bam2fq -a - | gzip > interleaved_reads.fq.gz
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updated by Ram Ram • written by madkitty madkitty

thanks, I tried, but so far I didn't find a right way to deinterleave the output fastq, or at least the script, which I tried was not correct.

I'm sure that samtools ended before I started to use bedtools, but I try again.

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written by anon • anon

2 answers

I know this post is old but I had the same issue. I was wondering if my data were paired-end or single-end. To do so I tried Bedtools but the resulting files were empty. So, I tried Picard with the paired-end command and it turned out that all my reads went to the unpaired file. Therefore my dataset was single-end and that's probably why the Bedtools command wouldn't work.

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written by mari.felix5723 • mari.felix5723

Please cite your command when you mention one in your solution, so that future users can see and better understand. (picard-tools : which command ? which flags ?)

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written by mariakondili13 • mariakondili13

I had the same issue as well. Just found out that the problem stems from sorting. I skipped the sorting step and directly convert the unsorted bam file into fastq file. It worked!

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written by gohchengang1994 • gohchengang1994

I tried what you suggested, it is still empty...

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written by w19oody • w19oody

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