[miso-users] Install miso using pip and mixed read length BAMs

[Maurits Evers]

Dear all.

I have been trying to install&run miso on my Mac and have run into a 
couple of problems/issues. Any help and/or clarifications would be 
greatly appreciated.

1. I did a global install following the recommended installation method 
using pip. Everything seems to install fine, and importing misopy and 
pysplicing from within python works. However, miso, module_availability 
and test_miso are unknown commands. Chasing the binaries on my machine, 
I can see that they are located at 
/opt/local/Library/Frameworks/Python.framework/Versions/2.7/bin. Adding 
this location to PATH fixes the issue of the unknown miso executables. 
Do I need to add anything else?

2. As to testing the install, module_availability runs fine. test_miso 
returns a "Run 0 tests in 0.000s". When I try to execute test_miso from 
within
/opt/local/Library/Frameworks/Python.framework/Versions/2.7/lib/python2.7/site-packages/misopy 
via python test_miso.py it seems to run the 3 tests mentioned in the 
documention, but I end up with errors such as the following

     .Testing conversion of SAM to BAM...
     Executing: sam_to_bam --convert
 
/opt/local/Library/Frameworks/Python.framework/Versions/2.7/lib/python2.7/site-packages/misopy/test-data/sam-data/c2c12.Atp2b1.sam
 
/opt/local/Library/Frameworks/Python.framework/Versions/2.7/lib/python2.7/site-packages/misopy/test-output/sam-output
     Converting SAM to BAM...
     Traceback (most recent call last):
        File
 
"/opt/local/Library/Frameworks/Python.framework/Versions/2.7/bin/sam_to_bam",
     line 9, in <module>
          load_entry_point('misopy==0.5.2', 'console_scripts',
     'sam_to_bam')()
        File
 
"/opt/local/Library/Frameworks/Python.framework/Versions/2.7/lib/python2.7/site-packages/misopy/sam_to_bam.py",
     line 63, in main
          sam_to_bam(sam_filename, output_dir, header_ref=ref)
        File
 
"/opt/local/Library/Frameworks/Python.framework/Versions/2.7/lib/python2.7/site-packages/misopy/sam_to_bam.py",
     line 13, in sam_to_bam
          os.makedirs(output_dir)
        File
 
"/opt/local/Library/Frameworks/Python.framework/Versions/2.7/lib/python2.7/os.py",
     line 150, in makedirs
          makedirs(head, mode)
        File
 
"/opt/local/Library/Frameworks/Python.framework/Versions/2.7/lib/python2.7/os.py",
     line 157, in makedirs
          mkdir(name, mode)
     OSError: [Errno 13] Permission denied:
 
'/opt/local/Library/Frameworks/Python.framework/Versions/2.7/lib/python2.7/site-packages/misopy/test-output'

I don't know why test_miso fails to run properly. Is this something to 
worry about?

3. I have paired-end mouse RNA-seq data which I mapped to the mm10 
reference genome using tophat. The bam file is sorted and indexed, and I 
indexed successfully the gff annotation file. Upon running miso with

     miso --run indexed ../alignment/tophat/WT.bam --settings-filename
     miso_settings.txt --output-dir WT/ --paired-end 472 277 --read-len 120

I get the warning that miso found mixed length reads within the BAM 
file. Prior to mapping, reads were adapter-trimmed and quality-filtered 
so naturally aligned reads will have a read-length distribution. I don't 
understand what to make of this warning. I would assume that most 
RNA-seq data consists of different read lengths, due to some form of 
trimming/filtering of the raw data. I don't understand why miso would 
require reads to have the same length in order to be able to estimate 
isoform expression. Could you advise how to proceed? The read length 
distribution shows reads with lengths between 20 and 120 nt. Running 
miso for each of the read lengths separately would be possible but 
tedious, requiring 100 separate runs followed by merging the individual 
output files.

Best regards,
Maurits


-- 
Dr. Maurits Evers
Center for Integrative Bioinformatics Vienna
Max F. Perutz Laboratories
Dr. Bohr Gasse 9
A-1030 Vienna, Austria



-- 
Dr. Maurits Evers
Statistical Bioinformatics
Institute of Functional Genomics
University of Regensburg
Josef-Engert-Str. 9 (Biopark I)
93053 Regensburg, Germany