10X

This repository contains scripts used to 10x pre-processing and RNA-velocity estimation with StarSolo.

Requirements

STAR >=2.7.3a
cellranger

If you don't have STAR available as a module you can install it with conda:

conda install -c bioconda star=2.7.3a

Velocity estimation with StarSolo

1. Find genome (fasta) and annotation (gtf) used with cellranger. You will need to rebuild the star index with STAR >=2.7.3a. Adjust the number of threads (--runThreadN) to your setup.

mkdir ./star
STAR \
  --runMode genomeGenerate \
  --genomeDir ./star/ \
  --genomeFastaFiles mm38.fa \
  --sjdbGTFfile mm38.gtf \
  --runThreadN 20

2. Run helper script for StarSolo

This script will check the necessary requirements and downloads the proper whitelist based on 10X chemistry. It also adjusted the parameters for StarSolo accordingly.

• -i path to fastq files
• -c 10X chemistry used v1, v2 or v3
• -s path to star index from step 1
• -t number of threads used
• last parameter is the output path

sh scripts/velocity.sh -i /path/to/fastq -c v3 -s /path/to/star -t 20 ./velocity

Notes

• reference preparation

Custom reference preparation

wget http://ftp.ensembl.org/pub/release-98/gtf/mus_musculus/Mus_musculus.GRCm38.98.chr.gtf.gz
wget http://ftp.ensembl.org/pub/release-98/fasta/mus_musculus/dna/Mus_musculus.GRCm38.dna.chromosome.{1..19,X,Y,MT}.fa.gz
gunzip -c Mus_musculus.GRCm38.dna.chromosome.*.fa.gz > mm38.fa
gunzip *.gt.gz > mm38.gtf

Credits

• Kevin Blighe: 10X chemistry
• Kevin Lane: template setup