[miso-users] error with miso run

[Yarden Katz]

Hi Fan,

A few questions on this:

1. Is your BAM file sorted and indexed, with the index (which in this case should be called "010_accepted_hits.bam") in the directory "010_bam/", i.e. in the same directory of the BAM?  If not, the call will fail.

2. Do your chromosome headers match?  Note that "Homo_sapiens.GRCh37.65.min_1000.const_exons.gff" is Ensembl derived, so it will contain Ensembl style chromosome headers (e.g. "1", "2", "3" for chromosomes 1, 2, 3, respectively), while if your genome index for Tophat/Bowtie was made with UCSC, it will contain the "chr" prefix.  If that's the case, none of your reads will map by "tagBam" and the call will fail.  See:

http://genes.mit.edu/burgelab/miso/docs/#answer4
http://genes.mit.edu/burgelab/miso/docs/#human-mouse-gene-models-for-isoform-centric-analyses

3. Finally, try executing:

tagBam -i /home/fan/Meningiomas_RNAseq/010_bam/010_accepted_hits.bam -files /home/fan/Downloads/exon_hg19/Homo_sapiens.GRCh37.65.min_1000.const_exons.gff -labels gff -intervals -f 1 | samtools view -h - | egrep '^@|:gff:'

If this call comes out empty, it's likely issue #2, a mismatch of headers.

Best, --Yarden

On Jan 29, 2013, at 3:03 PM, Fan Gao wrote:

> Dear MISO community,
> 
> I am a freshman to this software. Based on installation instruction, I installed MISO on my laptop that has been pre-installed with Ubuntu. I also installed bedtools and samtools as required.
> 
> I got the following error message when I tried to compute insert length. The bam file I used was from tophat.
> Does anyone know what is the problem?
> 
> Thanks a lot.
> 
> Fan
> ***************************************************************************************************************
> fan at fan-X45U:~/Meningiomas_RNAseq$ python ~fan/yarden-MISO-4f32ca4/misopy/pe_utils.py --compute-insert-len 010_bam/010_accepted_hits.bam /home/fan/Downloads/exon_hg19/Homo_sapiens.GRCh37.65.min_1000.const_exons.gff --output-dir 010_insert-dist/
> Computing insert length distribution of 1 files:
>  /home/fan/Meningiomas_RNAseq/010_bam/010_accepted_hits.bam
>  - Using const. exons from: /home/fan/Downloads/exon_hg19/Homo_sapiens.GRCh37.65.min_1000.const_exons.gff
>  - Outputting to: /home/fan/Meningiomas_RNAseq/010_insert-dist
>  - Minimum exon size used: 500
> Making directory: /home/fan/Meningiomas_RNAseq/010_insert-dist
> Getting constitutive exons...
>  - Input GFF: /home/fan/Downloads/exon_hg19/Homo_sapiens.GRCh37.65.min_1000.const_exons.gff
>  - Output dir: /home/fan/Meningiomas_RNAseq/010_insert-dist
>  - Output format: gff
>  - Including only exons greater than or equal to 500-bp
> Constitutive exons GFF was given, so not outputting another one.
> Filtering BAM reads
> Fetching reads in constitutive exons
> Mapping BAM to GFF...
>  - BAM: /home/fan/Meningiomas_RNAseq/010_bam/010_accepted_hits.bam
>  - GFF: /home/fan/Downloads/exon_hg19/Homo_sapiens.GRCh37.65.min_1000.const_exons.gff
>  - Output file: /home/fan/Meningiomas_RNAseq/010_insert-dist/bam2gff_Homo_sapiens.GRCh37.65.min_1000.const_exons.gff/010_accepted_hits.bam
> Preparing to call bedtools 'tagBam'
> tagBam -i /home/fan/Meningiomas_RNAseq/010_bam/010_accepted_hits.bam -files /home/fan/Downloads/exon_hg19/Homo_sapiens.GRCh37.65.min_1000.const_exons.gff -labels gff -intervals -f 1 | samtools view -h - | egrep '^@|:gff:' | samtools view -Shb -o /home/fan/Meningiomas_RNAseq/010_insert-dist/bam2gff_Homo_sapiens.GRCh37.65.min_1000.const_exons.gff/010_accepted_hits.bam - 
> [samopen] SAM header is present: 25 sequences.
> [sam_read1] reference 'ID:TopHat	CL:/home/fangao/tophat-2.0.4.Linux_x86_64/tophat -p 12 -o CGATGT_fusion_new2 --solexa1.3-quals --fusion-search --bowtie1 /home/fangao/seqlib/bowtie_index_hg19/hg19 s_1_1_CGATGT_sequence.txt s_1_2_CGATGT_sequence.txt	VN:2.0.4
> ' is recognized as '*'.
> [main_samview] truncated file.
> Traceback (most recent call last):
>  File "/home/fan/yarden-MISO-4f32ca4/misopy/pe_utils.py", line 528, in <module>
>    main()
>  File "/home/fan/yarden-MISO-4f32ca4/misopy/pe_utils.py", line 525, in main
>    sd_max=sd_max)
>  File "/home/fan/yarden-MISO-4f32ca4/misopy/pe_utils.py", line 272, in compute_insert_len
>    output_dir)
>  File "/usr/local/lib/python2.7/dist-packages/misopy/exon_utils.py", line 187, in map_bam2gff
>    raise Exception, "Error: tagBam call failed."
> Exception: Error: tagBam call failed.
> 
> **************************************************
> Fan Gao, Ph.D
> Research Associate
> Zilkha Neurogenetic Institute 
> Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research
> Keck School of Medicine
> University of Southern California
> 1425 San Pablo Street, BCC 409B
> Los Angeles, CA 90089
> United States
> Phone: 323-442-2497
> Email: gaof at usc.edu
> **************************************************
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