Question on BAM file

[dongfy88]

I used bowtie to generate *.sam file, then transform the *.sam file to *.bam and *.bam.bai (by samtools). Unfortunately, when I load *.bam file to IGB, nothing displays. The commands I used are :

bowtie -S e_coli reads/e_coli_10000snp.fq ec_snp.sam;
samtools view -bS -o ec_snp.bam ec_snp.sam;
samtools sort ec_snp.bam ec_snp.sorted;
samtools index ec_snp.sorted.sam;

IGB threw out an error on picard when loading the *bam file first time.

Also, I use tophat to generate *bam file, and got the same unavailable *bam file.

Does any one meet this problem before? Thank you in advance.

[gaffa]

By IGB I assume you mean IGV (Integrated Genome Browser).

What is the error message that you get?

[dongfy88]

By IGB I assume you mean IGV (Integrated Genome Browser).

What is the error message that you get?

Thank gaffa for your reply.
The error message is :
A SAMFormatException has been thrown by the Picard tools.
Please validate your BAM files and contact the Picard project at http://picard.sourceforge.net.See console for the details of the exception.

[12jrowley2]

Bam Error in IGB

Thank gaffa for your reply.
The error message is :
A SAMFormatException has been thrown by the Picard tools.
Please validate your BAM files and contact the Picard project at http://picard.sourceforge.net.See console for the details of the exception.

Yeah I also got the same error - processed my bowtie reads the same way. Any hints yet?

[d17]

A possible answer to your problems

bowtie -S e_coli reads/e_coli_10000snp.fq ec_snp.sam;
samtools view -bS -o ec_snp.bam ec_snp.sam;
samtools sort ec_snp.bam ec_snp.sorted;
samtools index ec_snp.sorted.sam;

If these are indeed the exact commands you used I noticed two errors:
1) In the second command you need to say -bhS to output the header to BAM
2) In the fourth command you should be indexing the BAM file not the SAM file

[12jrowley2]

Bam header formatting from bowtie incompatible with IGB

I do index the bam files.
I think you're right about the header being wrong, but using -bhS in this case does not solve the issue, because the @SQ lines are absent.

I tried adding the header as in:

http://samtools.sourceforge.net/samtools.shtml

samtools faidx ref.fa
samtools view -bt ref.fa.fai aln.sam > aln.bam

But this does not resolve the issue.
Although I have not tried it, the following suggestion of using picard may help:

http://permalink.gmane.org/gmane.science.biology.biodas/422

[feixue1039]

I do index the bam files.
I think you're right about the header being wrong, but using -bhS in this case does not solve the issue, because the @SQ lines are absent.

I tried adding the header as in:

http://samtools.sourceforge.net/samtools.shtml

samtools faidx ref.fa
samtools view -bt ref.fa.fai aln.sam > aln.bam

But this does not resolve the issue.
Although I have not tried it, the following suggestion of using picard may help:
http://permalink.gmane.org/gmane.sci...ogy.biodas/422

Hi,
I don't know how to solve your problem. Maybe you can have another try with the command line like this:
samtools faidx reference.fa
samtools import reference.fa.fai aligned_reads.sam aligned_reads.bam
samtools sort aligned_reads.bam aligned_reads_sorted
samtools index aligned_reads_sorted.bam
For more details,you can read the document below:

http://seqanswers.com/forums/showthread.php?t=7068

Hope it can help.

feixue1039

[dongfy88]

If these are indeed the exact commands you used I noticed two errors:
1) In the second command you need to say -bhS to output the header to BAM
2) In the fourth command you should be indexing the BAM file not the SAM file

the fourth command is BAM not SAM, I spelt it wrong here.
Actually, I have use picard to generate BAM file and index BAM file and passed the validation test, but when I loaded the BAM file, IGB still display nothing. And, this time, IGB did not throw out error.

[drdna]

IGB has some major issues. I have a bam file that was generated by tophat. First time I uploaded it into IGB, it displayed fine. However, when I tried loading a chromosome reference and a gtf against which to compare the alignments, the .bam file no longer displayed. Quite honestly, I don't like IGB at all, it is clunky, not intuitive and almost impossible to use. Plus, it has strange behaviors that make no sense at all. For example when I tried to change the reference sequence, it made the new "reference" a track instead. The only way I could figure out how to change the reference, while maintaining the existing tracks was to quit the session and start over. Very frustrating.