Bam2FastQ help

[Zapages]

Hi guys,

I am new here. I am trying to use Bam2Fastq convert a very large (~7 GB) bam file to a fastq file, but whenever I try to do this, I get this weird message.

Basically I am trying to extract only aligned reads from a bam file, using this site as my guide: http://davetang.org/wiki/tiki-index....ls#Basic_usage

If that is not possible. If it is possible to convert the whole file to fastq will be good as well.

What I enter in Command Prompt

bam2fastq -o blah_aligned.fastq --no-unaligned Bamfilename.bam

The message I get:

The sequences in the BAM file are marked as Paired, but a single output
file is specified.  Ensure that the output filename (--output) includes
a # symbol to be replaced with the read number

If you could kindly help. I will really appreciate it. Thank you.

-Zapages

[kmcarr]

Run the command

bam2fastq -o blah_aligned_R#.fastq --no-unaligned Bamfilename.bam

This will create two output files blah_aligned_R1.fastq and blah_aligned_R2.fastq containing read #1 and read #2 of the read pairs respectively.

[swbarnes2]

Yeah, the program is trying to do the right thing in giving you two separate fastq files, but you told it only one fastq, and it doesn't like that.

If you want only aligned reads, use samtools view -bF 4 to filter your .bam first.

[Zapages]

Thank you guys^

Yeah, the program is trying to do the right thing in giving you two separate fastq files, but you told it only one fastq, and it doesn't like that.

If you want only aligned reads, use samtools view -bF 4 to filter your .bam first.

I have used bamview and Bamseek to see the sequence that we are interested in are present. Everything lines up with the sequence that we are interested.

Anyway should I sort with samtools. I think the bam file is already aligned, but I am not too sure.

How do you sort in with sam tools?

[maubp]

Yeah, the program is trying to do the right thing in giving you two separate fastq files, but you told it only one fastq, and it doesn't like that.

That's rather frustrating - two paired FASTQ files is only the 'right thing' for some tools, others prefer a single interleaved FASTQ file (which makes piping streams much easier!).