Mouse_strain_genomeprep_snpsplit

GRCm38 and GRCm39 strain-specific reference genome generated with snp-split followed by sequence extraction based on chromosomal coordinates Run on GRCm38 to extract ref/cast/nmask sequences at ChIP-Seq peaks (rather than realign old data)
Run on GRCm39 to extract ref/cast/nmask sequences at Nucleoporin genes for genome-editing design

Summarry

The following commands were run to download appropriate GRCm38 and GRCm39 genomes and then to generate Castenous (Cast_EiJ) strain-specific genomes. While this largely followed the snpsplit genome gen guide (see references) there were some specific issues getting the backdated GRCm38 to work. This was run July 26th 2023 on the PITT HTC.

Obtain GRCm38 and GRCm39 genomes and place them in separate directories

wget -O Mus_musculus.GRCm39.dna.toplevel.fa.gz https://ftp.ensembl.org/pub/release-109/fasta/mus_musculus/dna/Mus_musculus.GRCm39.dna.toplevel.fa.gz & wget -c -O Mus_musculus.GRCm38.68.dna.toplevel.fa.gz ftp://ftp.ensembl.org/pub/release-68/fasta/mus_musculus/dna/Mus_musculus.GRCm38.68.dna.toplevel.fa.gz The GRCm38.68 ref was required for the v5 SNP build (other builds did not work), I could not access via the sanger.ac.uk link from legacy SNPsplit forum. Then unzip each within their folder.

Obtain GRCm38 and m39 strain specific SNPs in VCF format in another directory

wget -O mgp_REL2021_snps.vcf.gz ftp://ftp.sra.ebi.ac.uk/vol1/ERZ840/ERZ8406694/mgp_REL2021_snps.vcf.gz & wget -O mgp.v5.merged.snps_all.dbSNP142.vcf.gz https://ftp.ebi.ac.uk/pub/databases/mousegenomes/REL-1505-SNPs_Indels/mgp.v5.merged.snps_all.dbSNP142.vcf.gz &

Run SNPsplit_genome_preparation using SLURM

Run the Bash scripts CastEiJ_snpsplit_genomegen_GRCm39.bash and CastEiJ_snpsplit_genomegen_GRCm38_v68 including output for strain-specific genome

Combine chr.fa files for CAST and NMASK

From within the output Cast and Nmask directories run the following interactive commands:
cat *.fa > Mus_musculus.GRCm38.68.dna.CASTEiJ.fa &
cat *.fa > Mus_musculus.GRCm38.68.dna.NMASK.fa &

Extraction of Genomic Sequences of regions of interestr of Reference, N-masked and CastEiJ using Samtools

Generate .tsv with 2 columns (seqname and coordinates) and no header; manually typed out For GRCm38_v68 coordinates run CastEiJ_Samtools_NUP107peakseq_GRCm38_v68.bash which utilizes Nup107_mm10peak_GRCm38_v68_imprint.txt as input .tsv

Final step was to align in Clustal Omega for each sequence triad (ref/cast/nmask); easiest just to copy paste into web portal

https://www.ebi.ac.uk/Tools/msa/clustalo/

References and Links

https://felixkrueger.github.io/SNPsplit/genome_prep/legacy/ https://ftp.ebi.ac.uk/pub/databases/mousegenomes/ https://www.sanger.ac.uk/data/mouse-genomes-project/ https://www.mousegenomes.org/snps-indels/