Troubleshooting

I will put Frequently asked question here and provide solutions. Please refer to the Github Issue page for all the issues.

1. Singularity Error

If you encounter problem like below:

Current folder is /run
BAM file folder is bam
/usr/src/app/AltAnalyze.sh: line 25: cd: bam: No such file or directory

That is because there’s a folder called /run in your singularity sandbox, so the first line of AltAnalyze.sh which use relative path to enter /run folder will mistakenly go into the root folder, in turn cause the bam folder not found. The solution is go to your altanalyze/ sandbox, in /usr/src/app folder edit the AltAnalyze.sh first line from cd /run to cd /usr/src/app/run. Now the problem should be solved.

2. AltAnalyze warnings and errors

You can ignore messages shown below, it won’t affect your program.

Something like that:

library lxml not supported. WikiPathways and LineageProfiler visualization will not work. Please install with pip install lxml.
Traceback (most recent call last):
File "/usr/src/app/altanalyze/import_scripts/BAMtoJunctionBED.py", line 217, in parseJunctionEntries
    try: splicesite_db,chromosomes_found, gene_coord_db = retreiveAllKnownSpliceSites()
File "/usr/src/app/altanalyze/import_scripts/BAMtoJunctionBED.py", line 133, in retreiveAllKnownSpliceSites
    for file in os.listdir(parent_dir):
OSError: [Errno 2] No such file or directory: 'S'

Or:

AltAnalyze depedency not met for: patsy

...Combat batch effects correction requires pandas and patsy

AltAnalyze depedency not met for: wx

...The AltAnalyze Results Viewer requires wx

AltAnalyze depedency not met for: fastcluster

...Faster hierarchical cluster not supported without fastcluster

AltAnalyze depedency not met for: lxml

...Wikipathways visualization not supported without lxml



WARNING!!!! Some dependencies are not currently met.

This may impact AltAnalyze's performance

3. How to do HLA genotyping?

Here we provide one solution using Optitype, which has been on top-performing HLA genotyping tool for a long time. The author provide a docker container, I usually run like that:

# build image using singularity
singularity build my_software.sif docker://fred2/optitype

# run it, assuming the fastq file is in the current directory
sample='TCGA-XX-XXXX-1'
singularity run -B $(pwd):/mnt my_software.sif -i ${sample}.1.fastq ${sample}.2.fastq --rna -v -o /mnt

But what if you only have bam files, no worreis, just convert them to fastq first:

# this step is required, have to sort by chromsome name
samtools sort -n $BAM -o $SAMPLE.qsort.bam
# then just convert, more please look at bedtools bamtofastq docs, super easy
bedtools bamtofastq -i $SAMPLE.qsort.bam -fq $SAMPLE.1.fastq -fq2 $SAMPLE.2.fastq

After that, the result as a tsv file will be generated (30min probably), you can write your own parsing script for post-processing. In addition, this process can be parallelized, I often write it into a shell function, and use linux parallel tool to run like 20 jobs at the same time to speed thing up.