SIP is built based on Nextflow framework, thus requiring POSIX-compatible operating system (Linux, UNIX, MacOS X, or Windows subsystem for Linux). Apart form the OS requirements, SIP needs Java environment and Java version should be 1.8 or above.
SIP supports analyzing both under local environment and in docker containers. In the local mode, users should pre-install all needed bioinformatics tools and configure the environment variables properly so that these tools are available at command lines. In the container mode, users should download the docker image we prepared and set up a container so that SIP can execute analysis commands with tools prepared in the container image.
- Cell Home Directory: directory holding all raw sequencing fastq files
- Output Directory: final analysis results are placed here
- Working directory: intermediate results are placed here
Enter the working directory, run the following command to load Nextflow framework from internet.
curl -s https://get.nextflow.io | bashDownload SIP's code in the Working directory:
git clone https://github.com/chansigit/SIP.gitPlease put all data to be analyzed in the Cell Home Directory, and arrange them in the following way:
Suppose the path to Cell Home Directory is /data/TestDataset1 , users need to create multiple subfolders under /data/TestDataset1 and name them after each cell's identifier.
Each subfolder contains the raw sequencing fastq file(s) of each cell. For single-end experiments, the fastq file's name should be {CellName}.fastq; while for paired-end experiments, the fastq file pairs should be named as {CellName}_1.fastq and {CellName}_2.fastq.
As a summary, the arrangement of reads shoule be /data/TestDataset1/CellName/Cell1Name.fastq for a single-end experiment; and /data/TestDataset1/CellName/Cell1Name_1.fastq , /data/TestDataset1/CellName/Cell1Name_2.fastq for a paired-end experiment.
We choose fastp as the read quality control tool, salmon as the gene-expression quantification tool, and use read count as the quantification unit. We used the Seurat package for downstream analysis after quantification.
./nextflow run SIP.nf -resume \
--readqc=fastp --quant=salmon --quantunit=count \
--downstream=seurat \
--cellhome={Cell Home Directory Path} \
--output={Output Directory Path}