Troubleshoot - dgaolab/IRFinder GitHub Wiki

Issue 1: Empty reference files in the IRFinder folder

This could be due to the following problem:

  1. If you're using your own GTF that is not derived from Ensembl, make sure it contains the attribution tags transcript_biotype and gene_biotype, or their alternatives (supported since v1.2.3) transcript_type and gene_type. in the last column. IRFinder needs them to define intronic regions.
  2. Make sure your bedtools version is above 2.20. Older versions of bedtools miss some key functions required by IRFinder and some output formats are differentiated.
  3. Also check the number of chromosomes in your reference FASTA file. IRFinder opens the temporary files as many as the amounts of chromosomes at the same time during the reference preparation stage. However, there is a limitation of how many files can be opened at once, depending on the system setup (check by ulimit -n). If the chromosome number was more than the system allowance, the IRFinder would fail to generate IR reference. You will need root permission to increase the system limit.

Issue 2: Error Message during the quantification stage

Your error message might or might not look like the below one:

... version `GLIBCXX_xxx' not found ....

To fix this, first check your GCC version by gcc --version and GLIBC version by ldd --version and make sure they meet the minimal system requirements. Then try the following:

Step 1: You can cd into src/irfinder in the IRFinder package folder and recompile IRFinder by make clean; make. If you can successfully compile without error, it should generate irfinder binary file in the folder. You need to replace the older version by cp irfinder ../../bin/util/ and then go to Step 2.

If you encountered any problem, it's an indication of GCC/GLIBC incompatibility. Please first refer to the Compilation Session to see if it helps. Otherwise, please see how to contact us.

Step 2: After Step 1, cd into bin/util/ under IRFinder package folder. Then make a folder for testing purpose by mkdir test. Make sure your BAM file is sorted by names instead of coordinates. And try to run the following:

gzip -cd $BAMFILE | irfinder test $REF/IRFinder/ref-cover.bed $REF/IRFinder/ref-sj.ref $REF/IRFinder/ref-read-continues.ref $REF/IRFinder/ref-ROI.bed test/unsorted.frag.bam >> test/irfinder.stdout 2>> test/irfinder.stderr

You'll need to replace $BAMFILE with the full path to your input BAM file (unsorted) and replace $REF with the full path to your IRFinder reference folder. This command is supposed to run the quantification directly using the binary core instead of the IRFinder wrapper, which will expose the true error message instead of a vague segamentation fault. And it will save the output under the folder test, which you can remove later. The execution might take a while. If you encountered any problem, it's an indication of GLIBC incompatibility and please see how to contact us.

Issue 3: No Read Mapped in the BAM Mode

Please make sure your IR reference and your BAM file using the same format of chromosome identifier. Please note, Ensembl labels chromosomes as 1,2,3,....,X,Y while UCSC labels chromosomes as chr1,chr2,chr3,...chrX,chrY. The two are not compatible.