The RNA workflow consists of the following steps:
Tapestri RNA Pipeline similar to DNA starts with running a quality check (QC) module, which checks the validity of the input data. This QC module ensures that the FASTQ files used are valid and have no issues that might cause RNA Pipeline to fail.
The first step in the RNA workflow is similar to DNA pipeline where the adapters are trimmed and the RNA specific barcodes are extracted to obtain the RNA amplicon specific sequence from the reads.
This step outputs FASTQ files with barcodes in the read IDs, along with simple metrics that record the number of reads associated with each barcode observed in the FASTQ files.
- Step 4 - Genome alignment
Trimmed RNA reads are aligned to the provided reference genome using the STAR aligner [Dobin, A., Davis, C.A., Schlesinger, F., Drenkow, J., Zaleski, C., Jha, S., Batut, P., Chaisson, M. and Gingeras, T.R., 2013. STAR: ultrafast universal RNA-seq aligner. Bioinformatics, 29(1), pp.15-21.].
- Step 5 - Intronic reads filtering
Reads mapping to intronic regions, which is defined by the gaps between exons in the exon.bed file, are filtered out. The Pipeline employs a default threshold where any read containing 5 or more bases of intronic alignment is discarded.
- Step 6 - Read count generation
Then the reads aligned to specific RNA amplicons are counted for each barcode and added to the rna assay in the h5 file.