[miso-users] Error: tagBam call failed when running pe_utils.py --compute-insert-len

[Yarden Katz]

Hi,

See replies below:

On Jul 7, 2013, at 2:04 PM, Gu Mi wrote:

> Dear All:
> 
> I am using MISO to test for differential exon usage between a control and a treatment group. I got an error when computing the insert length distribution using pe_utils.py --compute-insert-len. I list the steps I used below:
> 
> 1. sort the BAM file from TopHat (by coordinate):
> samtools sort control.bam control_sorted
> 
> 2. index the BAM file:
> samtools index control_sorted.bam control_sorted.bai
> 
> 3. run pe_utils.py:
> python pe_utils.py --compute-insert-len controlam /directories/exons/Homo_sapiens.GRCh37.65.min_1000.const_exons.gff --output-dir /directories/insert-dist/
> 
> After the command above, I got the error message:
> 
> Preparing to call bedtools 'tagBam'
> tagBam -i control.bam -files /directories/exons/Homo_sapiens.GRCh37.65.min_1000.const_exons.gff -labels gff -intervals -f 1 | samtools view - -h | egrep '^@|:gff:' | samtools view - -Shb -o /directories/insert-dist/bam2gff_Homo_sapiens.GRCh37.65.min_1000.const_exons.gff/control.bam
> [samopen] SAM header is present: 25 sequences.
> [sam_read1] reference 'ID:TopHat        CL:/informatics/tools/Linux-AS5/bin/tophat -o Lane3 -g 1 --coverage-search --microexon -r 100 --phred64-quals --library-type fr-unstranded -p 4 -G gene_models/Homo_sapiens.GRCh37.72_norm.gtf --transcriptome-index=gene_models/transcripts /directories/Genomes/NCBI_Jul-09-2012/Human/bowtie/human_ref_genome Lane3_1.fq.gz Lane3_2.fq.gz     VN:1.4.1
> ' is recognized as '*'.
> [main_samview] truncated file.
> Traceback (most recent call last):
>   File "/pe_utils.py", line 520, in <module>
>     main()
>   File "pe_utils.py", line 517, in main
>     sd_max=sd_max)
>   File "pe_utils.py", line 271, in compute_insert_len
>     output_dir)
>   File "exon_utils.py", line 185, in map_bam2gff
>     raise Exception, "Error: tagBam call failed."
> Exception: Error: tagBam call failed.

It sounds like tagBam could not map any of your reads to the GFF, which is usually a headers mismatch issue.  

Could you confirm that your BAM file contains Ensembl style headers, and not UCSC ones?  What is the output of this command command for you:

samtools view -H /directories/insert-dist/bam2gff_Homo_sapiens.GRCh37.65.min_1000.const_exons.gff/control.bam

Also, what is the output of the following for you?

tagBam -i control.bam -files /directories/exons/Homo_sapiens.GRCh37.65.min_1000.const_exons.gff -labels gff -intervals -f 1 | samtools view - -h | egrep '^@|:gff:'

If this yields something empty, it means that tagBam could not match your BAM to the GFF, probably because of the chromosome headers mismatch issue that I mentioned.  

Best, --Yarden

> 
> I used Homo_sapiens.GRCh37.72_norm.gtf from Ensembl as the annotation file when preparing my data, but downloaded 
> 	• Human genome (hg19) alternative events v2.0
> from the MISO website and unzipped. I saw it is based on Homo_sapiens.GRCh37.65. Is this the version problem? If so, could anyone provide the latest GFF3 file for use? Thank you for your suggestions!
> 
> Best,
> Gu
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