Questions about genome preparations

[liyangyang12]

Hi Felix,
SNPsplit. what an amazing tool!
Recently, I took over an analysis project in which the reads from RNA-seq are needed to be splited into paternal and maternal ones (In the experiment we used female C57BL/6N and male PWK-PhJ). So, could you help me to check the genome preparation process? And what mean of C57BL_6NJ in mgp_REL2021_snps.vcf.gz? Should I add --strain2 C57BL_6NJ in my command (in my experiment , we used C57BL/6N rather than C57BL/6J)?
#genome preparations
SNPsplit_genome_preparation --strain PWK_PhJ --vcf /work/home/ackmmlgkeq/workspace3/SNP/mgp_REL2021_snps.vcf.gz --reference_genome /work/home/ackmmlgkeq/workspace3/genome/

[FelixKrueger]

Thanks for the kind words, and apologies for the late reply...

If I remember correctly the different variants of C57BL very closely related, so the command you suggested above should be absolutely fine. Edit: I just ran the genome preparation for C57BL_6NJ, and here is the number of SNPs:

13074   SNP were homozygous and passed high confidence filters and were thus included into the C57BL_6NJ genome

[FelixKrueger]

As opposed to:

20,235,830 SNP were homozygous and passed high confidence filters and were thus included into the PWK_PhJ genome

[liyangyang12]

Thank you very much !

[liyangyang12]

Hi Felix,
After running bismark_genome_preparation, I found that the SNP sites in “all_PWK_PhJ_SNPs_C57BL_6NJ_reference.based_on_GRCm39.txt” only include those from three chromosomes: chr1, chr2, and chr3. My command is "SNPsplit_genome_preparation --strain C57BL_6NJ --strain2 PWK_PhJ --vcf /work/home/ackmmlgkeq/workspace3/SNP/mgp_REL2021_snps.vcf.gz --reference_genome /work/home/ackmmlgkeq/workspace3/genome/ "，Why does this issue occur?

[FelixKrueger]

This is highly unusual, I really have no idea why this would be the case. Let me run the same command over here.

[FelixKrueger]

Just checking quickly, when you launch it, does it something like this:

Summarising SNPsplit Genome Preparation Parameters
==================================================
Processing SNPs from VCF file:          /Mus_musculus/MouseGenomeProject/mgp_REL2021_snps.vcf.gz
Reading/filtering VCF file:             Yes (default)
Reference genome:                        ...
N-masking:                              Yes
Full SNP genome:                        Yes
SNP strain:                             C57BL_6NJ
SNP strain 2:                           PWK_PhJ
Dual hybrid, new Ref/SNP:               C57BL_6NJ/PWK_PhJ

Using the following chromosomes (detected from VCF file >>/weka/projects/references/sync_structure/genomes/Mus_musculus/MouseGenomeProject/mgp_REL2021_snps.vcf.gz<<):
3       6       16      5       8       18      11      9       2       13      1       10      14      19      15      7       17      X       12      4

[FelixKrueger]

This works perfectly normal on my end:

Summary
20194355 Ns were newly introduced into the N-masked genome for strain/strain 2 [C57BL_6NJ/PWK_PhJ] in total
20194355 SNPs were newly introduced into the full sequence genome version for strainstrain 2 [C57BL_6NJ/PWK_PhJ] in total

[liyangyang12]

Thank you for your reply！ I will check if there are any issues with my vcf file.

[liyangyang12]

I have another question. I merged all the .fa files from folder “C57BL_6NJ_PWK_PhJ_dual_hybrid.based_on_GRCm39_N-masked” like this, and then used Bismark to create an index for the merged .fa file. Is this approach reasonable?

[FelixKrueger]

Catting the sequences is possible, but bot required (Bismark works with individual chromosome files, well as multi-FastA files). Pick one of the 2 options.

[liyangyang12]

Thank you very much!