The Transcript-level Quantification feature of Blast2GO allows quantifying the gene and isoform expression of RNA-seq datasets.
This video shows step-by-step how to create a count table of aligned sequencing reads and explains in detail the different concepts of expression quantification at transcript level. The application is based on the RSEM software package, which assigns reads to the isoforms they came from modelling the uncertainty derived from multiple isoforms having overlapping sequences.
As input, sequencing reads in FASTQ format and a FASTA file containing the transcript sequences are required. The output, an un-normalized count table, can then be analysed directly within Blast2GO. Various options for differential expression analysis are available (find videos here and here).
Find more details in the online user manual.
- Li B and Dewey CN (2011). “RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome.” BMC Bioinformatics, 12:323.
- Langmead B, Salzberg S (2012). “Fast gapped-read alignment with Bowtie 2.” Nature Methods, 9:357-359