Hi,
I'm running bam-readcount (commit 6c3f3ae901) on a few hundred bam files against a single reference fasta file (designated with -f). However, when I look at the output for any of the files, all of the reference bases, for any position, are N.
NODE_14_length_46_cov_1.239130 52 N 1 =:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00 A:1:2.00:36.00:2.00:1:0:0.00:0.02:0.00:0:0.00:0.00:0.00 C:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00 G:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00 T:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00 N:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00
Is this a bug? The base in the reference sequence at this position is A.
>NODE_14_length_46_cov_1.239130
AGCTAACTGAGTTTATCACACTCAGTTAATGTCCATTTCACTTCACACATAACCTTACAG
ATCGGAAGATCTCGTA
Thanks!
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Chris F. •
Yup - reference bases are being reported now. Thanks!
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Chris F. •
The only way I was able to reproduce this type of problem was by manually changing the reference fasta in a way that invalidates the index.
Assuming you have samtools handy, what is the result of:
samtools faidx ref.fa NODE_14_length_46_cov_1.239130:50-52
where ref.fa is your reference fasta? If you get something unexpected, try removing the "ref.fa.fai" file (or whatever yours is called) and running bam-readcount again (it will be automatically recreated).
One way this could happen is if you generated the fasta, ran bam-readcount, then regenerated the fasta with different contents. The fasta index would be stale in this case.
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tgi.tabbott
~/data7/src/samtools/samtools faidx contigs.fa NODE_14_length_46_cov_1.239130:50-52
>NODE_14_length_46_cov_1.239130:50-52
TAA
I blew away the index and recreated it with bam-readcount (it's the same size). Still getting N for the reference base.
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Chris F. •
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A total guess, but is there any chance the reference fasta file is wrong (i.e. not the one that was used to make the bams)? I seem to remember having a similar problem when I mixed up reference files doing similar things.
Thanks, but in this case, there's only a single reference file.