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Required files

Code from this repository

To clone the repository, type into your terminal:

git clone https://github.com/bioDS/phyloRNAanalysis

If you do not have git installed, type first:

conda install git

Now type:

cd phyloRNAanalysis

to enter the phyloRNAanalysis folder.

Reference genome

First, create reference folder in the phyloRNAanalysis directory:

mkdir reference
cd reference

Now, download and unzip the GRCh38v15 reference genome together with the associated index file:

wget ftp.ncbi.nlm.nih.gov/genomes/all/GCA/000/001/405/GCA_000001405.15_GRCh38/seqs_for_alignment_pipelines.ucsc_ids/GCA_000001405.15_GRCh38_no_alt_analysis_set.fna.gz
wget ftp.ncbi.nlm.nih.gov/genomes/all/GCA/000/001/405/GCA_000001405.15_GRCh38/seqs_for_alignment_pipelines.ucsc_ids/GCA_000001405.15_GRCh38_no_alt_analysis_set.fna.fai
gunzip GCA_000001405.15_GRCh38_no_alt_analysis_set.fna.gz

download and unzip the annotation:

wget ftp.ncbi.nlm.nih.gov/genomes/all/GCA/000/001/405/GCA_000001405.15_GRCh38/seqs_for_alignment_pipelines.ucsc_ids/GCA_000001405.15_GRCh38_full_analysis_set.refseq_annotation.gtf.gz
gunzip GCA_000001405.15_GRCh38_full_analysis_set.refseq_annotation.gtf.gz

as well as set of common variants (no need to unzip these):

wget ftp.ncbi.nlm.nih.gov/snp/organisms/human_9606/VCF/00-All.vcf.gz

Filter annotation file:

There is just one more thing. The newest version of Cellranger (version 5) does not accept an annotation file with extra scaffolds. This can be fixed by filtering the GTF file according to the fasta index file using the phyloRNA package:

Rscript -e 'phyloRNA::filter_gtf("GCA_000001405.15_GRCh38_full_analysis_set.refseq_annotation.gtf", "GCA_000001405.15_GRCh38_no_alt_analysis_set.fna.fai")'

Data

To download the GSE163210 data, navigate back to the phyloRNAanalysis, create a data directory and download the BAM files:

cd ..
mkdir data