RNA Editor fails at indexing BAM file step

[AliSajid]

Hi,

I am trying to develop a pipeline including your RNA Editor in it. However, when I try to use it, I run into an error. The software quits when it is at the indexing bam file step.

I have tried running it manually and samtools complains about the bamfile not being sorted or being corrupt.

The log file is as follows:

` INFO: CHECK DEPENDENCIES openjdk version "1.8.0_222" OpenJDK Runtime Environment (build 1.8.0_222-b10) OpenJDK 64-Bit Server VM (build 25.222-b10, mixed mode) INFO: Dependencies satisfied INFO: Start RnaEditor with: INFO: FastQ-File_1: ./SRR3438555_1.fastq INFO: FastQ-File_2: ./SRR3438555_2.fastq INFO: outfilePrefix:./rnaEditor/SRR3438555_1/SRR3438555_1 INFO: refGenome:/users/PJS0313/asimami/scratch/GRCH38/Homo_sapiens.GRCh38.dna.primary_assembly.fa INFO: dbsnp:/users/PJS0313/asimami/scratch/GRCH38/dbSNP.vcf INFO: sourceDir:/users/PJS0313/asimami/.local/bin/ INFO: threads:20 INFO: maxDiff:0.04 INFO: seedDiff:2 INFO: paired:True INFO: keepTemp:True INFO: overwrite:False INFO: INFO: [Mon Oct 14 12:16:39 2019] Align first Reads with BWA [DONE] Duration [0:09:08.359643] INFO: [Mon Oct 14 12:25:47 2019] Align second Reads with BWA [DONE] Duration [0:09:01.090889] INFO: [Mon Oct 14 12:34:48 2019] convert sai to sam [DONE] Duration [0:59:19.445871] INFO: [Mon Oct 14 13:34:08 2019] Sort Bam File [DONE] Duration [0:18:01.413584] INFO: [Mon Oct 14 13:52:09 2019] Index Bam File None

Traceback (most recent call last): File "/users/PJS0313/asimami/scratch/RNAEditor-master/RNAEditor.py", line 75, in run self.startAnalysis() File "/users/PJS0313/asimami/scratch/RNAEditor-master/RNAEditor.py", line 98, in startAnalysis mapResultFile=self.mapFastQ.startAnalysis() File "/users/PJS0313/asimami/scratch/RNAEditor-master/MapFastq.py", line 137, in startAnalysis Helper.proceedCommand("Index Bam File", cmd, samFile, bamFile+".bai", self.rnaEdit) File "/users/PJS0313/asimami/scratch/RNAEditor-master/Helper.py", line 286, in proceedCommand Helper.error(description+ " failed!!!",logFile,textField) File "/users/PJS0313/asimami/scratch/RNAEditor-master/Helper.py", line 753, in error raise Exception("\n\n" + Helper.prefix + "ERROR: " + message + Helper.praefix + "\n\n") Exception:

ERROR: Index Bam File failed!!!

Traceback (most recent call last): File "/users/PJS0313/asimami/scratch/RNAEditor-master/RNAEditor.py", line 77, in run Helper.error("RnaEditor Failed",self.logFile,self.textField) File "/users/PJS0313/asimami/scratch/RNAEditor-master/Helper.py", line 753, in error raise Exception("\n\n" + Helper.prefix + "ERROR: " + message + Helper.praefix + "\n\n") Exception:

ERROR: RnaEditor Failed `

Can you help with this?

[djhn75]

This sound like a problem with Samtools. Could you check if the sorted Bam file was created properly?

[AliSajid]

Checking using the various tricks including https://github.com/IARCbioinfo/BAM-tricks#check-if-bam-file-is-sorted shows me that it was indeed sorted correctly.

It appears that samtools is unable to support BAI indices for large genomes. I am filing a request there as well. Meanwhile, if samtools indeed has trouble, the workflow will need to be adjusted here too. Will keep you updated.