When importing RNA-Seq FASTQ files into Subio Platform, a pipeline consisting of fastp, HISAT2, and StringTie is used to calculate gene expression levels. No workstation or advanced UNIX command-line skills are required, and the processing can be performed on a standard Windows or Mac computer.
Before running the pipeline, prepare the required tools, HISAT2 index, and GTF file. For setup instructions, see the page corresponding to your operating system:
Operation Guide – Importing RNA-Seq FASTQ Files
Keep the FASTQ files in .gz compressed format. Because many intermediate files are generated in the same folder during processing, make sure that sufficient disk space is available (at least five times the total size of the FASTQ files). If the disk runs out of space during processing, the pipeline will stop. The FASTQ files may also be stored on an external drive.
Do not include spaces or non-ASCII characters in the paths to the FASTQ files or other files used by the pipeline.
When importing FASTQ files from paired-end samples, follow the required file-naming rules.
If the pipeline does not run successfully, first try asking ChatGPT or another AI assistant about the error message and your settings. If you are still unable to resolve the problem, you can also use our FASTQ Processing Troubleshooting Service .
After importing the FASTQ files, you may also find the following tutorial helpful: “RNA-Seq Gene Expression Data Analysis”.