Error: tagBam call failed when running pe_utils.py --compute-insert-len

[alittleboy]

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.

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!

[space_monkey]

I'm actually having the same problem with MISO. I also haven't been able to find anything as to why this happens, so if anyone could give us some insight, that would be great.

For now I'm trying to do the same thing using Bowtie and Picard-tools (outlined here: http://vinaykmittal.blogspot.ca/2012...or-paired.html ) but I'm sure it would be much easier using MISO's function...

[alittleboy]

I'm actually having the same problem with MISO. I also haven't been able to find anything as to why this happens, so if anyone could give us some insight, that would be great.

For now I'm trying to do the same thing using Bowtie and Picard-tools (outlined here: http://vinaykmittal.blogspot.ca/2012...or-paired.html ) but I'm sure it would be much easier using MISO's function...

Hi @space_monkey:

Here are some comments -- I contact the authors and gave my partial outputs, and the reply is below:

It does look like a headers mismatch then. Your BAM file contains "chr" style chromosomes (e.g. "chr10" and not "10"). I believe your GFF, /directories/exons/Homo_sapiens.GRCh37.65.min_1000.const_exons.gff, is from Ensembl which would not contain chr-prefixes. Just look in that gff file and see what the chromosome entries are like. If they don't have chr, the operation will fail. All you need to do is generate a constitutive exons file from a UCSC gff which contains chromosome headers that match your .bam file.

See:

http://genes.mit.edu/burgelab/miso/docs/#human-mouse-gene-models-for-isoform-centric-analyses

If you're using hg19, you can use this GFF:

http://genes.mit.edu/burgelab/miso/annotations/ucsc_tables/hg19/ensGene.gff3

Use our exon_utils program to generate constitutive exons from this file and then rerun pe_utils with that, instead of the GRCh37 gff file.

I use ensGene.gff3 and it works. However, I still cannot get the results (not sure why...)