Reference-Free Analysis of Long-Read RNA Sequencing for Non-Model Species
Fabian Jetzinger, Doctoral Candidate at BioBam Bioinformatics, explains how to characterize RNA isoforms from long-read data without a reference genome, using the isON-pipeline in OmicsBox.
Isoform identification tools such as FLAIR and IsoQuant work well when a reliable reference genome and transcriptome are available, but offer limited use for non-model species. The isON-pipeline, developed by Kristoffer Sahlin (LongTREC supervisor) (Stockholm University), fills this gap by reconstructing transcript isoforms directly from long reads, for both Oxford Nanopore (ONT) and PacBio data.
In this article, Fabian walks through the three tools of the isON-pipeline, shows how to run them in OmicsBox and explains how to interpret and build on the results.
What You Will Learn
What You Will Learn
- isONclust: How similar reads are grouped into clusters without a reference, even when different genes share similarity or a single gene produces several isoforms.
- isONcorrect: How sequencing errors in ONT reads are corrected by comparing them against a consensus built from similar reads in the same cluster.
- isONform: How clustered reads are resolved into distinct transcript isoform sequences.
- Running the pipeline in OmicsBox: Importing pre-processed FASTQ reads, selecting the data type (ONT or PacBio) and tuning parameters to obtain a FASTA file of isoforms and a table of supporting read counts per sample.
- Downstream analysis: Exploring isoform length and read-support distributions, annotating transcripts with the Functional Analysis module and testing differential expression with edgeR, NOISeq or maSigPro.
Why This Matters
Reference genomes and transcriptomes remain the preferred option when they exist, but many organisms still lack them. The reference-free isON-pipeline opens long-read transcriptomics to these species, enabling isoform-level analyses, and potentially new discoveries, in organisms that were previously out of reach.
📖 Read the full post here: https://www.biobam.com/reference-free-analysis-of-long-read-rna-sequencing-for-non-model-species/
Reference genomes and transcriptomes remain the preferred option when they exist, but many organisms still lack them. The reference-free isON-pipeline opens long-read transcriptomics to these species, enabling isoform-level analyses, and potentially new discoveries, in organisms that were previously out of reach.
📖 Read the full post here: https://www.biobam.com/reference-free-analysis-of-long-read-rna-sequencing-for-non-model-species/
