Value Error for chromosome number

[swethas112]

Hi,

I am trying to use IsoQuant for my Nanopore data. But, I keep getting the following ValueError. I wonder if there is some issue with the annotation file.

Here is the command I had used:
isoquant.py -d nanopore --fastq WT.fastq.gz KO.fastq.gz --reference hg38.fa --genedb gencode.v45.basic.annotation.gtf --complete_genedb --count_exons --output isoquant_new

Traceback (most recent call last):
  File "/home/ssubramanian/.conda/envs/isoquant/lib/python3.8/concurrent/futures/process.py", line 239, in _process_worker
    r = call_item.fn(*call_item.args, **call_item.kwargs)
  File "/home/ssubramanian/.conda/envs/isoquant/lib/python3.8/concurrent/futures/process.py", line 198, in _process_chunk
    return [fn(*args) for args in chunk]
  File "/home/ssubramanian/.conda/envs/isoquant/lib/python3.8/concurrent/futures/process.py", line 198, in <listcomp>
    return [fn(*args) for args in chunk]
  File "/home/ssubramanian/.conda/envs/isoquant/share/isoquant-3.6.3-0/src/dataset_processor.py", line 141, in collect_reads_in_parallel
    for gene_info, assignment_storage in alignment_collector.process():
  File "/home/ssubramanian/.conda/envs/isoquant/share/isoquant-3.6.3-0/src/alignment_processor.py", line 260, in process
    for res in self.forward_alignments(alignment_storage):
  File "/home/ssubramanian/.conda/envs/isoquant/share/isoquant-3.6.3-0/src/alignment_processor.py", line 279, in forward_alignments
    yield self.process_alignments_in_region(new_region, alignments)
  File "/home/ssubramanian/.conda/envs/isoquant/share/isoquant-3.6.3-0/src/alignment_processor.py", line 285, in process_alignments_in_region
    assignment_storage = self.process_intergenic(alignment_storage, current_region)
  File "/home/ssubramanian/.conda/envs/isoquant/share/isoquant-3.6.3-0/src/alignment_processor.py", line 294, in process_intergenic
    corrector = IlluminaExonCorrector(self.chr_id, region[0], region[1], self.illumina_bam)
  File "/home/ssubramanian/.conda/envs/isoquant/share/isoquant-3.6.3-0/src/illumina_exon_corrector.py", line 38, in __init__
    self.short_introns, self.counts = self.get_introns(short_read_file, chromosome, start, end)
  File "/home/ssubramanian/.conda/envs/isoquant/share/isoquant-3.6.3-0/src/illumina_exon_corrector.py", line 56, in get_introns
    intr = samfile.find_introns(samfile.fetch(chromosome, start = start, stop = end))
  File "pysam/libcalignmentfile.pyx", line 1092, in pysam.libcalignmentfile.AlignmentFile.fetch
  File "pysam/libchtslib.pyx", line 683, in pysam.libchtslib.HTSFile.parse_region
ValueError: invalid contig `chr1`
"""

The above exception was the direct cause of the following exception:

Traceback (most recent call last):
  File "/home/ssubramanian/.conda/envs/isoquant/bin/isoquant.py", line 819, in <module>
    main(sys.argv[1:])
  File "/home/ssubramanian/.conda/envs/isoquant/bin/isoquant.py", line 813, in main
    run_pipeline(args)
  File "/home/ssubramanian/.conda/envs/isoquant/bin/isoquant.py", line 763, in run_pipeline
    dataset_processor.process_all_samples(args.input_data)
  File "/home/ssubramanian/.conda/envs/isoquant/share/isoquant-3.6.3-0/src/dataset_processor.py", line 456, in process_all_samples
    self.process_sample(sample)
  File "/home/ssubramanian/.conda/envs/isoquant/share/isoquant-3.6.3-0/src/dataset_processor.py", line 480, in process_sample
    self.collect_reads(sample)
  File "/home/ssubramanian/.conda/envs/isoquant/share/isoquant-3.6.3-0/src/dataset_processor.py", line 558, in collect_reads
    for read_groups, alignment_stats, processed_reads in results:
  File "/home/ssubramanian/.conda/envs/isoquant/lib/python3.8/concurrent/futures/process.py", line 484, in _chain_from_iterable_of_lists
    for element in iterable:
  File "/home/ssubramanian/.conda/envs/isoquant/lib/python3.8/concurrent/futures/_base.py", line 619, in result_iterator
    yield fs.pop().result()
  File "/home/ssubramanian/.conda/envs/isoquant/lib/python3.8/concurrent/futures/_base.py", line 437, in result
    return self.__get_result()
  File "/home/ssubramanian/.conda/envs/isoquant/lib/python3.8/concurrent/futures/_base.py", line 389, in __get_result
    raise self._exception
ValueError: invalid contig `chr1` ```

Thanks in advance!

[andrewprzh]

Dear @swethas112

Seems like a problem with the reference genome and annotation, are you sure chromosome have the same name in hg38.fa and gencode.v45.basic.annotation.gtf ?

Best
Andrey

[swethas112]

Hi @andrewprzh ,

Yes, I checked that already.

[andrewprzh]

Looks really odd since the exception is caused by pysam lib, not IsoQuant itself, could you send the entire log file?

[swethas112]

Sure.

isoquant.log

[andrewprzh]

Something is particularly odd is with chr1, all other chromosomes are processed normally.
Could you send me the BAM files header (samtools view -H)?
Also, you can try to remove .fai index file if it was created before IsoQuant run and rerun IsoQuant.

Best
Andrey

[swethas112]

Here is the header from one of the BAM files

header.txt

I'll try after removing the index file

[andrewprzh]

BAM file looks fine, really puzzling...

[swethas112]

I tried it again after removing the .fai file, but I still get the same error