how to extract raw unaligned reads?

[joseph]

Hello

I used bowtie to map sequencing reads:

> bowtie ~/hg19 -v 2 -k 5 --best --strata -S -t reads.fastq reads.sam
End-to-end 2/3-mismatch full-index search: 01:00:21
# reads processed: 12084153
# reads with at least one reported alignment: 9391748 (77.72%)
# reads that failed to align: 2692405 (22.28%)
Reported 30293838 alignments to 1 output stream(s)

I would like to make a fastq file from the reads with at least one reported alignment [(9391748 (77.72%)].
Converting sam to bam and then using the samtools view with the -F 4 option gives me the Reported 30293838 alignments.
Any suggestions will be appreciated.
Joseph

[Nicolas]

You can use Bowtie's options (--al and --un) to output a fast[aq] files with the unmapped reads, and/or as fast[aq] with the reads that align at least once:

--al <filename>
Write all reads for which at least one alignment was reported to a file with name <filename>. Written reads will appear as they did in the input, without any of the trimming or translation of quality values that may have taken place within bowtie. Paired-end reads will be written to two parallel files with _1 and _2 inserted in the filename, e.g., if <filename> is aligned.fq, the #1 and #2 mates that fail to align will be written to aligned_1.fq and aligned_2.fq respectively.

--un <filename>
Write all reads that could not be aligned to a file with name <filename>. Written reads will appear as they did in the input, without any of the trimming or translation of quality values that may have taken place within Bowtie. Paired-end reads will be written to two parallel files with _1 and _2 inserted in the filename, e.g., if <filename> is unaligned.fq, the #1 and #2 mates that fail to align will be written to unaligned_1.fq and unaligned_2.fq respectively. Unless --max is also specified, reads with a number of valid alignments exceeding the limit set with the -m option are also written to <filename>.

I hope it helps

[joseph]

Originally Posted by joseph

Hello

I used bowtie to map sequencing reads:

> bowtie ~/hg19 -v 2 -k 5 --best --strata -S -t reads.fastq reads.sam
End-to-end 2/3-mismatch full-index search: 01:00:21
# reads processed: 12084153
# reads with at least one reported alignment: 9391748 (77.72%)
# reads that failed to align: 2692405 (22.28%)
Reported 30293838 alignments to 1 output stream(s)

I would like to make a fastq file from the reads with at least one reported alignment [(9391748 (77.72%)].
Converting sam to bam and then using the samtools view with the -F 4 option gives me the Reported 30293838 alignments.
Any suggestions will be appreciated.
Joseph

thank you for your help.