Required soft and versions:

Prerequisites as input:

1. RNA-seq data for F1-cross, fastq file (single end or paired end)

For example, rep_i.fastq for each of i={1..N} replicates.

2. Reference genome

For example, GRCm38_68.fa.

wget ftp://ftp-mouse.sanger.ac.uk/ref/GRCm38_68.fa

3. gtf annotation for the reference genome

For example, Mus_musculus.GRCm38.68.gtf.gz for corresponding reference genome version.

wget ftp://ftp.ensembl.org/pub/release-68/gtf/mus_musculus/Mus_musculus.GRCm38.68.gtf.gz
gunzip Mus_musculus.GRCm38.68.gtf.gz

4. Two vcf files for maternal and paternal imbred line (should be "compatible" with reference genome)

For example, 129S1_SvImJ.mgp.v5.snps.dbSNP142.vcf.gz and CAST_EiJ.mgp.v5.snps.dbSNP142.vcf.gz for 129S1 and CAST mice lines.

wget ftp://ftp-mouse.sanger.ac.uk/current_snps/strain_specific_vcfs/129S1_SvImJ.mgp.v5.snps.dbSNP142.vcf.gz
wget ftp://ftp-mouse.sanger.ac.uk/current_snps/strain_specific_vcfs/CAST_EiJ.mgp.v5.snps.dbSNP142.vcf.gz

or

Joint vcf file for multiple species, where two lines are presented (should be "compatible" with reference genome)

For example, mgp.v5.merged.snps_all.dbSNP142.vcf.gz for multiple mice lines.

wget ftp://ftp-mouse.sanger.ac.uk/current_snps/mgp.v5.merged.snps_all.dbSNP142.vcf.gz

Note: remember about vcf calling by mutect, varscan, etc

Input preprocessing:

1. Reference fasta should be indexed.

2. vcf-files should be bgzip-ed and indexed with tabix.

So the full list of files is:

Reference preparation:

prepare_reference.sh --ref GRCm38_68.fa --gtf Mus_musculus.GRCm38.68.gtf.gz \
  --name_mat 129S1_SvImJ --name_pat CAST_EiJ --vcf_mat 129S1_SvImJ.mgp.v5.snps.dbSNP142.vcf.gz --vcf_pat CAST_EiJ.mgp.v5.snps.dbSNP142.vcf.gz \
  --o_dir *output_directory_path/*
prepare_reference.sh --ref GRCm38_68.fa --gtf Mus_musculus.GRCm38.68.gtf.gz \
  --name_mat 129S1_SvImJ --name_pat CAST_EiJ --vcf mgp.v5.merged.snps_all.dbSNP142.vcf.gz \
  --o_dir *output_directory_path/*

Note: If directories are not writable, will create all supporting files at output_directory_path/refereces/. If something is not provided, it will be also created at output_directory_path/refereces/ (i.e. will eat some extra place!).

• Creating pseudogenomes from reference genomes and vcf file(s)

• F1-cross vcf files

• Gene-Transcript-Exon Annotations

Pipeline-specific fastq aligning:

For different tools there is required an alignment with:

• Unique read placement

• Multiple read placement

You can either use the product of your own alignment method (read about the correct way in each pipeline section) or:

prepare_alignments.sh ...

Pipeline specific bam processing (from bam or fastq, all the steps above):

prepare_ASE_tables.sh ...

GTEX-style pipeline

QuASAR-MPRA

ASE-TIGAR

MutRSeq

RSEM

Kallisto (for check)

Replicates analysis: