单细胞实战 (2)：cellranger 使用

wkd=/home/project/single-cell/MCC

cat SRR_Acc_List-2586-4.txt |while read i

do

fastq-dump --gzip --split-files ${i}.sra

done

cat SRR_Acc_List-9245-3.txt | while read i

do

mv ${i}_1*.gz${i}_S1_L001_I1_001.fastq.gz

mv ${i}_2*.gz ${i}_S1_L001_R1_001.fastq.gz

mv ${i}_3*.gz ${i}_S1_L001_R2_001.fastq.gz

done

SRR7722937

├── SRR7722937_S1_L001_I1_001.fastq.gz

├── SRR7722937_S1_L001_R1_001.fastq.gz

└── SRR7722937_S1_L001_R2_001.fastq.gz

curl -o cellranger-3.1.0.tar.gz "https://cf.10xgenomics.com/releases/cell-exp/cellranger-3.1.0.tar.gz?Expires=1601922176&Policy=eyJTdGF0ZW1lbnQiOlt7IlJlc291cmNlIjoiaHR0cHM6Ly9jZi4xMHhnZW5vbWljcy5jb20vcmVsZWFzZXMvY2VsbC1leHAvY2VsbHJhbmdlci0zLjEuMC50YXIuZ3oiLCJDb25kaXRpb24iOnsiRGF0ZUxlc3NUaGFuIjp7IkFXUzpFcG9jaFRpbWUiOjE2MDE5MjIxNzZ9fX1dfQ__&Signature=mCC-emSQTqWg3i6Rm23lkuccNRI4z7xf-8bJ-O5gHqsYFJHDPZN9MeUZW5vhRZvmZhiGK7EDL4y~3xPtQbS6JtfUL9EFsoUxMywyF7tGN2ZlU2pbb2EVpmhKjmDWPVmvurDb~ZlHVYZCYcOc6gEHVtFRre-ICa7-nccVtnUJA-HvxNrZMs5mlQaUG9E-ngtbLi86gvIYlMHYnFRieZYpMA-kmUKrHGG8MhiuBGR96AWOrsVdTyMjD-BJOurGYvZGddWAF5uVXNPJs47FqF4fNCSQw71WOSRx4bQGdfb-jrOFp-NDSYFhkY1-gigku8VCx9phFCtyTAVM9yHeGN1oMQ__&Key-Pair-Id=APKAI7S6A5RYOXBWRPDA"

tar -zxvf cellranger-3.1.0.tar.gz

export PATH=/datadisk02/ScRNAseq/cellranger-3.1.0:$PATH

wget ftp://ftp.ensembl.org/pub/release-93/fasta/homo_sapiens/dna/Homo_sapiens.GRCh38.dna.primary_assembly.fa.gz

gunzip Homo_sapiens.GRCh38.dna.primary_assembly.fa.gz

wget ftp://ftp.ensembl.org/pub/release-93/gtf/homo_sapiens/Homo_sapiens.GRCh38.93.gtf.gz

gunzip Homo_sapiens.GRCh38.93.gtf.gz

cellranger mkgtf Homo_sapiens.GRCh38.93.gtf Homo_sapiens.GRCh38.93.filtered.gtf \

                 --attribute=gene_biotype:protein_coding \

                 --attribute=gene_biotype:lincRNA \

                 --attribute=gene_biotype:antisense \

                 --attribute=gene_biotype:IG_LV_gene \

                 --attribute=gene_biotype:IG_V_gene \

                 --attribute=gene_biotype:IG_V_pseudogene \

                 --attribute=gene_biotype:IG_D_gene \

                 --attribute=gene_biotype:IG_J_gene \

                 --attribute=gene_biotype:IG_J_pseudogene \

                 --attribute=gene_biotype:IG_C_gene \

                 --attribute=gene_biotype:IG_C_pseudogene \

                 --attribute=gene_biotype:TR_V_gene \

                 --attribute=gene_biotype:TR_V_pseudogene \

                 --attribute=gene_biotype:TR_D_gene \

                 --attribute=gene_biotype:TR_J_gene \

                 --attribute=gene_biotype:TR_J_pseudogene \

                 --attribute=gene_biotype:TR_C_gene

cellranger mkref --genome=GRCh38 \

                 --fasta=Homo_sapiens.GRCh38.dna.primary_assembly.fa \

                 --genes=Homo_sapiens.GRCh38.93.filtered.gtf \

                 --ref-version=3.0.0

curl -O https://cf.10xgenomics.com/supp/cell-exp/refdata-cellranger-GRCh38-3.0.0.tar.gz

cellranger count --id=SRR937 --transcriptome=GRCh38 --fastqs=SRR7722937/ --sample=SRR7722937

Generating STAR genome index (may take over 8 core hours for a 3Gb genome)...

15:12:38 ..... Started STAR run

15:12:38 ... Starting to generate Genome files

15:14:31 ... starting to sort  Suffix Array. This may take a long time...

15:14:41 ... sorting Suffix Array chunks and saving them to disk...