CellRanger Alignmnet

Author

Ahmed M. Elhossiny

CellRanger and Reference Genome Configuration

  • We aligned all the raw data using CellRanger (v7.1.0). We used the module installed on our HPC, but you can download as follows
curl -o cellranger-7.1.0.tar.gz "https://cf.10xgenomics.com/releases/cell-exp/cellranger-7.1.0.tar.gz?Expires=1761892950&Key-Pair-Id=APKAI7S6A5RYOXBWRPDA&Signature=Q-tZwrJgZZlHEk3xY05gLIX~q-ZhDFa7UXGD0BpAnHZuxJQfyzMTyhwY3C6aE3ARRQfVS5XEVTlGiMQRX8~cFF7VzafPjFQPCAQD2sZjb1p4hmot0SqVRp4jCdf~ISTdr~I9pKMsjjkx3DtGSQ6htXAqKJYocn8UTz5fOHkLNCIlBeW4jykdacAFRZJVkAJAKxf0dBAned37xA9liwnx1nqFKPREbqpIdRW1e8wdSQiO4P50ODrnqMZ23q0Ylp7e7uqQoIIxD1~Nf6OcwwSg8ZLNnyZbyuMDB7CB74sad1SLyMQL9N2mMaZhg6krDsN001ATHWk4epNMOs95DONDKw__"

Installation instructions can be found detailed here

  • We used GRCh38 reference genome that can be downloaded as follows
# Create directory
mkdir -p ../data/references
refDir=../data/references

# Download reference file
curl --output ../data/references/refdata-gex-GRCh38-2020-A.tar.gz \
https://cf.10xgenomics.com/supp/cell-exp/refdata-gex-GRCh38-2020-A.tar.gz

# Uncompress zipped file
tar -xvf ../data/references/refdata-gex-GRCh38-2020-A.tar.gz \
-C ../data/references/
rm ../data/references/refdata-gex-GRCh38-2020-A.tar.gz

Running CellRanger

The following shell script is submitted to a high-performance computing (HPC) cluster as a SLURM job array to process each of the samples in parallel.

The script pulls a cellranger_manifest.txt file from data directory with at two columns: sample_id and cellranger_output path for each sample as follow

sample_1 path/to/sample_1/fastq
sample_2 path/to/sample_2/fastq
sample_3 path/to/sample_3/fastq 
#!/bin/bash

#SBATCH --account=
#SBATCH --job-name='cellranger_hg38_%a'
#SBATCH --output=logs/cellranger_hg38_%a.log

#SBATCH --partition=standard
#SBATCH --mem=128G
#SBATCH --cpus-per-task=16
#SBATCH --time=48:00:00

#SBATCH --array=1-7

mkdir -p logs

# Import modules
ml cellranger/7.1.0

# Setting up variables
samples_manifest=../data/cellranger_manifest.txt
ref=../data/references/refdata-gex-GRCh38-2020-A
outputDir=../outputs/alignment
mkdir -p $outputDir

# Extracting sample for the current job 
current_sample_name=$(cat $samples_manifest | cut -f1 | sed -n ${SLURM_ARRAY_TASK_ID}p)
current_sample_path=$(cat $samples_manifest | cut -f2 | sed -n ${SLURM_ARRAY_TASK_ID}p)

echo "Analyzing Sample $current_sample_name" 
echo -e "Fastq path $current_sample_path"

# Running cellranger
cellranger count --id=${current_sample_name} \
--transcriptome=$ref \
--fastqs=$current_sample_path \
--sample=$current_sample_name \
--localcores=16

mv ${current_sample_name} $outputDir