Error on gemline calling

[carolwxm]

Hello, Thank you for developing such a wonderful tool. Recently, I attempted to execute somatic variant calling from scRNA-seq data. The preprocess and germline calling sections executed successfully. However, I encountered an issue during the first step of somatic calling.

My code: python {path}/Monopogen/src/Monopogen.py somatic \ -a {path}/Monopogen/apps \ -r region.lst -t 16 \ -i {path}/${sample} \ -l ./cell_barcode_annotations.tsv \ -s featureInfo \ -g {path}/GRCh38.fa

Error Message: [2023-10-26 10:05:38,384] INFO Monopogen.py Get feature information from sequencing data... [E::hts_open_format] Failed to open file "{output_path}/germline/chr7.phased.vcf.gz" : No such file or directory [E::idx_find_and_load] Could not retrieve index file for '{output_path}/germline/chr1.phased.vcf.gz' [W::vcf_parse] Contig 'chr1' is not defined in the header. (Quick workaround: index the file with tabix.) [E::idx_find_and_load] Could not retrieve index file for '/{output_path}/germline/chr2.phased.vcf.gz' [W::vcf_parse] Contig 'chr2' is not defined in the header. (Quick workaround: index the file with tabix.) [E::idx_find_and_load] Could not retrieve index file for '{output_path}/germline/chr4.phased.vcf.gz' [W::vcf_parse] Contig 'chr4' is not defined in the header. (Quick workaround: index the file with tabix.) [E::idx_find_and_load] Could not retrieve index file for '{output_path}/germline/chr3.phased.vcf.gz' [W::vcf_parse] Contig 'chr3' is not defined in the header. (Quick workaround: index the file with tabix.) [E::idx_find_and_load] Could not retrieve index file for '{path}/germline/chr5.phased.vcf.gz' [W::vcf_parse] Contig 'chr5' is not defined in the header. (Quick workaround: index the file with tabix.) [E::idx_find_and_load] Could not retrieve index file for '/{output_path}/germline/chr6.phased.vcf.gz' [W::vcf_parse] Contig 'chr6' is not defined in the header. (Quick workaround: index the file with tabix.) [E::idx_find_and_load] Could not retrieve index file for '{output_path}/germline/chr8.phased.vcf.gz' [W::vcf_parse] Contig 'chr8' is not defined in the header. (Quick workaround: index the file with tabix.) [E::idx_find_and_load] Could not retrieve index file for '{output_path}/germline/chr9.phased.vcf.gz' [W::vcf_parse] Contig 'chr9' is not defined in the header. (Quick workaround: index the file with tabix.) [E::idx_find_and_load] Could not retrieve index file for '{output_path}/germline/chr10.phased.vcf.gz' [W::vcf_parse] Contig 'chr10' is not defined in the header. (Quick workaround: index the file with tabix.) [E::idx_find_and_load] Could not retrieve index file for '{output_path}/germline/chr11.phased.vcf.gz' [W::vcf_parse] Contig 'chr11' is not defined in the header. (Quick workaround: index the file with tabix.) [E::idx_find_and_load] Could not retrieve index file for '{output_path}/germline/chr12.phased.vcf.gz' [W::vcf_parse] Contig 'chr12' is not defined in the header. (Quick workaround: index the file with tabix.) [E::idx_find_and_load] Could not retrieve index file for '{output_path}/germline/chr13.phased.vcf.gz' [W::vcf_parse] Contig 'chr13' is not defined in the header. (Quick workaround: index the file with tabix.) [E::idx_find_and_load] Could not retrieve index file for '{output_path}/germline/chr14.phased.vcf.gz' [W::vcf_parse] Contig 'chr14' is not defined in the header. (Quick workaround: index the file with tabix.) [E::idx_find_and_load] Could not retrieve index file for '{output_path}/germline/chr16.phased.vcf.gz' [W::vcf_parse] Contig 'chr16' is not defined in the header. (Quick workaround: index the file with tabix.) [E::idx_find_and_load] Could not retrieve index file for '{output_path}/germline/chr15.phased.vcf.gz' [W::vcf_parse] Contig 'chr15' is not defined in the header. (Quick workaround: index the file with tabix.) [E::idx_find_and_load] Could not retrieve index file for '{output_path}/germline/chr2.gl.vcf.gz' [E::idx_find_and_load] Could not retrieve index file for '{output_path}/germline/chr4.gl.vcf.gz' [E::idx_find_and_load] Could not retrieve index file for '{output_path}/germline/chr1.gl.vcf.gz' [E::idx_find_and_load] Could not retrieve index file for '{output_path}/germline/chr5.gl.vcf.gz' [E::idx_find_and_load] Could not retrieve index file for '{output_path}/germline/chr3.gl.vcf.gz' [E::idx_find_and_load] Could not retrieve index file for '{output_path}/germline/chr8.gl.vcf.gz' [E::idx_find_and_load] Could not retrieve index file for '{output_path}/germline/chr6.gl.vcf.gz' [E::idx_find_and_load] Could not retrieve index file for '{output_path}/germline/chr12.gl.vcf.gz' [E::idx_find_and_load] Could not retrieve index file for '{output_path}/germline/chr9.gl.vcf.gz' [E::idx_find_and_load] Could not retrieve index file for '{output_path}/germline/chr10.gl.vcf.gz' [E::idx_find_and_load] Could not retrieve index file for '{output_path}/germline/chr11.gl.vcf.gz' [E::idx_find_and_load] Could not retrieve index file for '{output_path}germline/chr13.gl.vcf.gz' [E::idx_find_and_load] Could not retrieve index file for '{output_path}/germline/chr14.gl.vcf.gz' [E::idx_find_and_load] Could not retrieve index file for '{output_path}/germline/chr17.phased.vcf.gz' [W::vcf_parse] Contig 'chr17' is not defined in the header. (Quick workaround: index the file with tabix.) [E::idx_find_and_load] Could not retrieve index file for '{output_path}/germline/chr16.gl.vcf.gz' [E::idx_find_and_load] Could not retrieve index file for '{output_path}/germline/chr15.gl.vcf.gz' [E::idx_find_and_load] Could not retrieve index file for '{output_path}/germline/chr17.gl.vcf.gz' [E::idx_find_and_load] Could not retrieve index file for '{output_path}/germline/chr18.phased.vcf.gz' [W::vcf_parse] Contig 'chr18' is not defined in the header. (Quick workaround: index the file with tabix.) [E::idx_find_and_load] Could not retrieve index file for '{output_path}/germline/chr18.gl.vcf.gz' [E::hts_open_format] Failed to open file "{output_path}/germline/chr19.phased.vcf.gz" : No such file or directory [E::idx_find_and_load] Could not retrieve index file for '{output_path}/germline/chr20.phased.vcf.gz' [W::vcf_parse] Contig 'chr20' is not defined in the header. (Quick workaround: index the file with tabix.) [E::idx_find_and_load] Could not retrieve index file for '{output_path}/germline/chr20.gl.vcf.gz' [E::idx_find_and_load] Could not retrieve index file for '{output_path}/germline/chr21.phased.vcf.gz' [W::vcf_parse] Contig 'chr21' is not defined in the header. (Quick workaround: index the file with tabix.) [E::idx_find_and_load] Could not retrieve index file for '{output_path}/germline/chr21.gl.vcf.gz' [E::idx_find_and_load] Could not retrieve index file for '{output_path}/germline/chr22.phased.vcf.gz' [W::vcf_parse] Contig 'chr22' is not defined in the header. (Quick workaround: index the file with tabix.) [E::idx_find_and_load] Could not retrieve index file for '{output_path}/germline/chr22.gl.vcf.gz' multiprocessing.pool.RemoteTraceback: """ Traceback (most recent call last): File "~/miniconda2/envs/Monopogen/lib/python3.8/multiprocessing/pool.py", line 125, in worker result = (True, func(*args, *kwds)) File "~/miniconda2/envs/Monopogen/lib/python3.8/multiprocessing/pool.py", line 48, in mapstar return list(map(args)) File "/Monopogen/src/somatic.py", line 88, in featureInfo vcf_in = VariantFile(out + "/germline/" + region + ".phased.vcf.gz") File "pysam/libcbcf.pyx", line 4117, in pysam.libcbcf.VariantFile.init File "pysam/libcbcf.pyx", line 4342, in pysam.libcbcf.VariantFile.open FileNotFoundError: [Errno 2] could not open variant file b'{output_path}/germline/chr7.phased.vcf.gz': No such file or directory """

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

Traceback (most recent call last): File "/Monopogen/src/Monopogen.py", line 435, in main() File "/Monopogen/src/Monopogen.py", line 428, in main args.func(args) File "/Monopogen/src/Monopogen.py", line 150, in somatic result = pool.map(featureInfo, joblst) File "~/miniconda2/envs/Monopogen/lib/python3.8/multiprocessing/pool.py", line 364, in map return self._map_async(func, iterable, mapstar, chunksize).get() File "~/miniconda2/envs/Monopogen/lib/python3.8/multiprocessing/pool.py", line 771, in get raise self._value FileNotFoundError: [Errno 2] could not open variant file b'{output_path}/germline/chr7.phased.vcf.gz': No such file or directory

I would be very grateful if you could provide some guidance on how to resolve this issue?

[jinzhuangdou]

It seems the germline module is not finished successfully. Are you able to examine whether there are variants called in chr7.phased.vcf.gz?