genome selection

[Hemantcnaik]

Hello, I am trying run your pipeline on single cell analysis, i have genome C57(maternal) CAST(Paternal) and CAST(maternal) C57(paternal) in this case how to use the command python3 python3/prepare_reference.py, i have SNP file downloaded from mouse genome project (CAST snp file)

[Strausyatina]

Hello, Looks like your case is Inbred lines: Separate line(s) vcf with one of them being the reference line itself. Please have a look at the tables in the corresponding sections Pseudoreference fasta creation and Heterozygous(parental) VCF creation for the parameters you need. Please also note the footnote "[1] If one or the alleles in case of inbred lines is reference, then everything should be provided as mat or pat only, consistently." That implies that in the first case you would need to provide your CAST snp file as --vcf_pat, and in the second as --vcf_mat.

[Hemantcnaik]

Hello thank you for your reply i have tried below command but i am getting error, can u please help me

command used python3 ASEReadCounter_star-master/python3/prepare_reference.py --PSEUDOREF True --HETVCF True \ --pseudoref_dir pseudo \ --vcf_dir vcf \ --ref Ref/Mus_musculus.GRCm38.68.dna.toplevel.fa \ --name_pat CAST_EiJ \ --vcf_pat CAST_EiJ.mgp.v5.snps.dbSNP142.vcf.gz \ --gtf Ref/Mus_musculus.GRCm38.99.chr.gtf

erorr I am getting /File "ASEReadCounter_star-master/python3/prepare_reference.py", line 718, in main() File "ASEReadCounter_star-master/python3/prepare_reference.py", line 603, in main GATK_SelectVariants(r=args.ref, v=args.vcf_mat, o=snp_alt_vcf.name, b=False) File "ASEReadCounter_star-master/python3/prepare_reference.py", line 79, in GATK_SelectVariants for item in flags[f]: TypeError: 'NoneType' object is not iterable

[Strausyatina]

Sorry for that, the freshly committed version, with this bug fixed, should work now.

One more thing, the procedure of creating a heterozygous VCF follows the rules below, in case of Inbred lines:

• if none of maternal or paternal strains is reference, the resulting heterozygous VCF will consider maternal variants as reference, and paternal variants as alternative
• if one of the strains is reference, the rest one would be automatically alternative

So you don't need to repeat this operation twice, heterozygous VCF will be the same. You should just remember which parent was actual or set reference, and which is alternative (for C57(maternal) vs CAST(Paternal): maternal is reference in the resulting count tables at the end of the pipeline, and paternal is alternative; reverse for CAST(maternal) vs C57(paternal)).

This therefore means that in python2/allelecounter.py function the corresponding reference --ref should be used: either maternal pseudoreference or reference genome itself respectively.

I should add this clarification to the wiki, thank you!

Please let me know if you have any other questions.

Asia

[Hemantcnaik]

Thank you for your clarification