GEXSCOPE Single cell analysis
Project description
CeleScope
GEXSCOPE Single Cell Analysis Tool Kit
中文文档
Requirements
- conda
- linux
- minimum 32GB RAM(to run STAR aligner)
Installation
git clone https://github.com/zhouyiqi91/CeleScope.git
cd CeleScope
source setup.sh
Reference genome
Homo sapiens
wget ftp://ftp.ensembl.org/pub/release-99/fasta/homo_sapiens/dna/Homo_sapiens.GRCh38.dna.primary_assembly.fa.gz
wget ftp://ftp.ensembl.org/pub/release-99/gtf/homo_sapiens/Homo_sapiens.GRCh38.99.gtf.gz
mkdir -p references/Homo_sapiens/Ensembl/GRCh38
gzip -c -d Homo_sapiens.GRCh38.dna.primary_assembly.fa.gz > references/Homo_sapiens/Ensembl/GRCh38/Homo_sapiens.GRCh38.fa
gzip -c -d Homo_sapiens.GRCh38.99.gtf.gz > references/Homo_sapiens/Ensembl/GRCh38/Homo_sapiens.GRCh38.99.gtf
conda activate celescope
gtfToGenePred -genePredExt -geneNameAsName2 references/Homo_sapiens/Ensembl/GRCh38/Homo_sapiens.GRCh38.99.gtf /dev/stdout | \
awk '{print $12"\t"$1"\t"$2"\t"$3"\t"$4"\t"$5"\t"$6"\t"$7"\t"$8"\t"$9"\t"$10}' > references/Homo_sapiens/Ensembl/GRCh38/Homo_sapiens.GRCh38.99.refFlat
STAR \
--runMode genomeGenerate \
--runThreadN 6 \
--genomeDir references/Homo_sapiens/Ensembl/GRCh38 \
--genomeFastaFiles references/Homo_sapiens/Ensembl/GRCh38/Homo_sapiens.GRCh38.fa \
--sjdbGTFfile references/Homo_sapiens/Ensembl/GRCh38/Homo_sapiens.GRCh38.99.gtf \
--sjdbOverhang 100
Mus musculus
wget ftp://ftp.ensembl.org/pub/release-99/fasta/mus_musculus/dna/Mus_musculus.GRCm38.dna.primary_assembly.fa.gz
wget ftp://ftp.ensembl.org/pub/release-99/gtf/mus_musculus/Mus_musculus.GRCm38.99.gtf.gz
mkdir -p references/Mus_musculus/Ensembl/GRCm38
gzip -c -d Mus_musculus.GRCm38.dna.primary_assembly.fa.gz > references/Mus_musculus/Ensembl/GRCm38/Mus_musculus.GRCm38.fa
gzip -c -d Mus_musculus.GRCm38.99.gtf.gz > references/Mus_musculus/Ensembl/GRCm38/Mus_musculus.GRCm38.99.gtf
conda activate celescope
gtfToGenePred -genePredExt -geneNameAsName2 references/Mus_musculus/Ensembl/GRCm38/Mus_musculus.GRCm38.99.gtf /dev/stdout | \
awk '{print $12"\t"$1"\t"$2"\t"$3"\t"$4"\t"$5"\t"$6"\t"$7"\t"$8"\t"$9"\t"$10}' > references/Mus_musculus/Ensembl/GRCm38/Mus_musculus.GRCm38.99.refFlat
STAR \
--runMode genomeGenerate \
--runThreadN 6 \
--genomeDir references/Mus_musculus/Ensembl/GRCm38 \
--genomeFastaFiles references/Mus_musculus/Ensembl/GRCm38/Mus_musculus.GRCm38.fa \
--sjdbGTFfile references/Mus_musculus/Ensembl/GRCm38/Mus_musculus.GRCm38.99.gtf \
--sjdbOverhang 100
Usage
Single cell RNA-Seq
conda activate celescope
celescope rna run\
--fq1 ./data/R2005212_L1_1.fq.gz\
--fq2 ./data/R2005212_L1_2.fq.gz\
--chemistry scopeV2.1.1\
--genomeDir /SGR/references/Homo_sapiens/Ensembl/GRCh38\
--sample R2005212\
--thread 4
--fq1
Required. gzipped FASTQ read 1 file path
--fq2
Required. gzipped FASTQ read 2 file path
--chemistry
Required. chemistry version, choices=['scopeV2.0.0', 'scopeV2.0.1', 'scopeV2.1.0', 'scopeV2.1.1']
--genomeDir
Required. reference genome directory path
--sample
Required. sample name
--thread
Required. number of threads
Single Cell VDJ
conda activate celescope
celescope vdj run\
--fq1 {vdj fq1.gz}\
--fq2 {vdj fq2.gz}\
--sample {sample name}\
--chemistry scopeV2.0.1\
--thread 4\
--type {TCR or BCR}
--match_dir {match_dir}\
--type
Required. TCR or BCR
--match_dir
Optional. Matched scRNA-Seq directory after running CeleScope
Single Cell Multiplexing
conda activate celescope
celescope smk run\
--fq1 {smk fq1.gz}\
--fq2 {smk fq2.gz}\
--sample {sample name}\
--chemistry scopeV2.0.1\
--SMK_pattern L25C45\
--SMK_barcode {SMK barcode fasta}\
--SMK_linker {SMK linker fasta}\
--match_dir {match_dir}\
--dim 2\
--combine_cluster {combine_cluster.tsv}
SMK_pattern
Required. L25C45 means 25 bp linker + 45 bp cell barcode
abbreviations:
C: cell barcode
U: UMI
T: polyT
L: linker
--SMK_barcode
Required. SMK tag fasta file
--SMK_linker
Required. SMK linker fasta file
--match_dir
Required. Matched scRNA-Seq directory after running CeleScope
--dim
Required. SMK dimension
--combine_cluster
Optional. Conbine cluster tsv file.
first column: original cluster number
second column: combined cluster number
$cat SMK_barcode.fasta
>SMK1
ATTCAAGGGCAGCCGCGTCACGATTGGATACGACTGTTGGACCGG
>SMK2
TGGATGGGATAAGTGCGTGATGGACCGAAGGGACCTCGTGGCCGG
>SMK3
CGGCTCGTGCTGCGTCGTCTCAAGTCCAGAAACTCCGTGTATCCT
>SMK4
ATTGGGAGGCTTTCGTACCGCTGCCGCCACCAGGTGATACCCGCT
>SMK5
CTCCCTGGTGTTCAATACCCGATGTGGTGGGCAGAATGTGGCTGG
>SMK6
TTACCCGCAGGAAGACGTATACCCCTCGTGCCAGGCGACCAATGC
$cat SMK_linker.fasta
>smk_linker
GTTGTCAAGATGCTACCGTTCAGAG
$cat combine_cluster.tsv
1 1
2 2
3 2
4 2
5 3
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