將SRA轉(zhuǎn)為fastq
數(shù)據(jù)下載好之后，我們得到的是SRR文件梳庆，需要將其轉(zhuǎn)換為fastq格式才能使用
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
結(jié)束之后每個(gè)SRR文件會(huì)變成解壓出三個(gè)文件
為什么會(huì)有三個(gè)文件，這個(gè)需要去查一下10X的官方說(shuō)明
可以看到Read1是26bp卑惜，主要是16bp的barcode和10bp的UMI膏执，Read2的長(zhǎng)度不固定，所以不一定是98bp露久，視情況而定更米，還有一個(gè)I7 index長(zhǎng)度是8bp
接下來(lái)就按照10X的標(biāo)準(zhǔn)去對(duì)文件進(jìn)行改名
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 ${i}_S1_L001_R2_001.fastq.gz
done
之后我創(chuàng)建了文件夾將每個(gè)SRR數(shù)據(jù)的三個(gè)fastq文件放到一個(gè)文件夾下
SRR7722937
├── SRR7722937_S1_L001_I1_001.fastq.gz
├── SRR7722937_S1_L001_R1_001.fastq.gz
└── SRR7722937_S1_L001_R2_001.fastq.gz
cellranger使用
下載cellranger3.1版本（目前cellranger已經(jīng)支持4.0版本）并添加至環(huán)境變量
curl -o cellranger-3.1.0.tar.gz "https://cf.10xgenomics.com/releases/cell-exp/cellranger-3.1.0.tar.gz?Expires=1601922176&Policy=eyJTdGF0ZW1lbnQiOlt7IlJlc291cmNlIjoiaHR0cHM6Ly9jZi4xMHhnZW5vbWljcy5jb20vcmVsZWFzZXMvY2VsbC1leHAvY2VsbHJhbmdlci0zLjEuMC50YXIuZ3oiLCJDb25kaXRpb24iOnsiRGF0ZUxlc3NUaGFuIjp7IkFXUzpFcG9jaFRpbWUiOjE2MDE5MjIxNzZ9fX1dfQ__&Signature=mCC-emSQTqWg3i6Rm23lkuccNRI4z7xf-8bJ-O5gHqsYFJHDPZN9MeUZW5vhRZvmZhiGK7EDL4y~3xPtQbS6JtfUL9EFsoUxMywyF7tGN2ZlU2pbb2EVpmhKjmDWPVmvurDb~ZlHVYZCYcOc6gEHVtFRre-ICa7-nccVtnUJA-HvxNrZMs5mlQaUG9E-ngtbLi86gvIYlMHYnFRieZYpMA-kmUKrHGG8MhiuBGR96AWOrsVdTyMjD-BJOurGYvZGddWAF5uVXNPJs47FqF4fNCSQw71WOSRx4bQGdfb-jrOFp-NDSYFhkY1-gigku8VCx9phFCtyTAVM9yHeGN1oMQ__&Key-Pair-Id=APKAI7S6A5RYOXBWRPDA"
tar -zxvf cellranger-3.1.0.tar.gz
export PATH=/datadisk02/ScRNAseq/cellranger-3.1.0:$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
也可以下載構(gòu)建好的注釋
curl -O https://cf.10xgenomics.com/supp/cell-exp/refdata-cellranger-GRCh38-3.0.0.tar.gz
然后進(jìn)行比對(duì)即可
cellranger count --id=SRR937 --transcriptome=GRCh38 --fastqs=SRR7722937/ --sample=SRR7722937
在分析過(guò)程中可以發(fā)現(xiàn)有些命令比較眼熟，不難發(fā)現(xiàn)cellranger的比對(duì)還是構(gòu)建索引其實(shí)都有STAR的影子毫痕，后續(xù)的話我將STARsolo（利用STAR分析單細(xì)胞數(shù)據(jù)）的流程再整理一下征峦。
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...
參考鏈接
10x_Technical_Note_Base_Composition_SC3_v2_RevB.pdf
【生信技能樹】使用10X單細(xì)胞轉(zhuǎn)錄組數(shù)據(jù)探索免疫治療
