The pipleline of single cell RNA

1 Alignment (fastq->bam)

(1) 10X Genomics fastq data

If we get fastq data from 10X Genomics, we can do alignment using cellranger with the parameter:

cellranger count \
--id=run_count_1kpbmcs \
--fastqs=/home/01.example_script/02.cellranger/pbmc_1k_v3_fastqs\
--sample=pbmc_1k_v3\
--transcriptome=/home/01.example_script/02.cellranger/refdata-gex-GRCh38-2020-A/

id means the output folder name .
fastqs means the folder which contains the input fastq files.
sample means sample id
transcriptome means reference file download from cellranger website.
Then we can get a output folder named run_count_1kpbmc. The files in the run_count_1kpbmc are as below:

图片.png
The files in the out folder are as below:
图片.png
we used the possorted_genome_bam.bamand filtered_feature_bc_matrix as input files for further analysis.
(2) non 10X Genomics fastq data
we can do aligment use the software hisat2 with parameter :

/hisat2 \
-q \
-x /02.Hisat2/01.prepare_data/grcm38/genome\
-U /03.downloaddata2-2022.7.27/01.fastq_dta/SRR3557021.fastq \
-S /03.downloaddata2-2022.7.27/02.hisat2_align_bam/SRR3557021/SRR3557021.sam\
samtools view \
-bS /03.downloaddata2-2022.7.27/02.hisat2_align_bam/SRR3557021/SRR3557021.sam > /nfs/public/liuyang/03.downloaddata2-2022.7.27/02.hisat2_align_bam/SRR3557021/SRR3557021.bam
/usr/bin/samtools sort /nfs/public/liuyang/03.downloaddata2-2022.7.27/02.hisat2_align_bam/SRR3557021/SRR3557021.bam -o /nfs/public/liuyang/03.downloaddata2-2022.7.27/02.hisat2_align_bam/SRR3557021/SRR3557021.sort.bam
rm -r /nfs/public/liuyang/03.downloaddata2-2022.7.27/02.hisat2_align_bam/SRR3557021/SRR3557021.sam