Aaron,

Thanks for sharing the new version for testing allelic imbalance. I'm starting to try to port over our WASP AI testing to WASP2. I thought I would start from .merge.bam files and recount those.

As background, I have VCF files which represent a simulated diploid genome. Previously the chr-separated VCF worked fine with WASP pipeline all the way to CHT, but there may be some issues with those VCF, where I need to add additional fields or tags.

As a side question, can I use the AI test in WASP2 with counts from WASP? E.g. these:

wasp_cht/alt_as_counts.sample_A_1.h5
wasp_cht/hap_read_counts.sample_A_1.adj
wasp_cht/hap_read_counts.sample_A_1.hetp
wasp_cht/hap_read_counts.sample_A_1.txt
wasp_cht/other_as_counts.sample_A_1.h5
wasp_cht/read_counts.sample_A_1.h5
wasp_cht/ref_as_counts.sample_A_1.h5

If not, with the WASP2 counting script, I am now getting this error:

python run_analysis.py count --rna -ft data/Drosophila_melanogaster.BDGP6.28.100.chr.gtf.gz \
  -a wasp_mapping/sample_A_1.merge.bam -g data/drosophila_wg.vcf -s sample \
  -r data/Drosophila_melanogaster.BDGP6.28.100.chr.gtf.gz -o testai
Namespace(command='count', stype='rna', singlecell=False, 
features=['data/Drosophila_melanogaster.BDGP6.28.100.chr.gtf.gz'], 
alignment='wasp_mapping/sample_A_1.merge.bam', 
genotypes='data/drosophila_wg.vcf', sample='sample', 
regions='data/Drosophila_melanogaster.BDGP6.28.100.chr.gtf.gz', 
barcodes=None, output='testai', nofilt=False, keeptemps=None)
Bulk Analysis
GTF filtered by feature
Filtering reads that overlap regions of interest
Bam file filtered!
Traceback (most recent call last):
  File "/proj/milovelab/bin/WASP2/src/analysis/run_analysis.py", line 273, in <module>
    main()
  File "/proj/milovelab/bin/WASP2/src/analysis/run_analysis.py", line 266, in main
    parse_counting(args.alignment, args.genotypes, args.regions, 
args.sample, args.output, args.stype, nofilt=args.nofilt, temp_loc=args.keeptemps, features=args.features)
  File "/proj/milovelab/bin/WASP2/src/analysis/run_analysis.py", line 53, in parse_counting
    intersect_df = preprocess_data(in_bam, in_vcf, in_region, in_sample, stype, nofilt, tmpdir, features)
  File "/proj/milovelab/bin/WASP2/src/analysis/run_analysis.py", line 34, in preprocess_data
    write_sample_snp(in_vcf, in_sample, out_dir)
  File "/proj/milovelab/bin/WASP2/src/analysis/filter_data.py", line 24, in write_sample_snp
    vcf = VariantFile(in_file)
  File "pysam/libcbcf.pyx", line 4054, in pysam.libcbcf.VariantFile.__init__
  File "pysam/libcbcf.pyx", line 4284, in pysam.libcbcf.VariantFile.open
ValueError: invalid file `b'data/drosophila_wg.vcf'` (mode=`b'r'`) - is it VCF/BCF format?

The VCF in question has some non-required fields missing but it appears valid:

vcf-validator data/drosophila_wg.vcf
Could not parse the fileformat version string [#CHROM	POS	ID	REF	ALT	QUAL	FILTER	INFO	FORMAT	sample], assuming VCFv4.2
The header tag 'reference' not present. (Not required but highly recommended.)
The "fileformat" field not present in the header, assuming VCFv4.2
The header tag 'contig' not present for CHROM=2L. (Not required but highly recommended.)
column sample at 2L:10591 .. FORMAT tag [GL] not listed in the header,FORMAT tag [GT] not listed in the header
The header tag 'contig' not present for CHROM=2R. (Not required but highly recommended.)
The header tag 'contig' not present for CHROM=3L. (Not required but highly recommended.)
The header tag 'contig' not present for CHROM=3R. (Not required but highly recommended.)
The header tag 'contig' not present for CHROM=4. (Not required but highly recommended.)
The header tag 'contig' not present for CHROM=X. (Not required but highly recommended.)

The VCF file looks like:

#CHROM	POS	ID	REF	ALT	QUAL	FILTER	INFO	FORMAT	sample
2L	10591	rs1	T	A	.	PASS	.	GT:GL	0|1:-100,0,-100
2L	11464	rs2	A	T	.	PASS	.	GT:GL	0|1:-100,0,-100
...

Hi

Thanks for your great tool.

I want to perform ASE calling from 10X scRNA-seq data. Is there an example file for barcodes file(barcodes and their group/cell mapping) so that I can follow the tutorial? Many thanks.

Best