celescope 1.1.3

GEXSCOPE Single cell analysis

CeleScope

Requirements

• conda
• linux
• minimum 32GB RAM(to run STAR aligner)

Installation

git clone https://github.com/zhouyiqi91/CeleScope.git
cd CeleScope
source build.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 scopeV2.0.1\
 --genomeDir /SGR/references/Homo_sapiens/Ensembl/GRCh38\
 --sample R2005212\
 --thread 4

--fq1 gzipped FASTQ read 1 file path
--fq2 gzipped FASTQ read 2 file path
--chemistry chemistry version, choices=['scopeV2.0.0', 'scopeV2.0.1', 'scopeV2.1.0', 'scopeV2.1.1']
--genomeDir reference genome directory path
--sample sample name
--thread number of threads

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