# Detection of Expressed Otx mRNA Isoforms in Sea Urchins by Mapping NGS Reads to Single-Gene/Transcript Sequences

## TL;DR

This paper shows how mapping NGS reads to single-gene sequences can detect mRNA isoforms in sea urchins, validated by RT-qPCR.

## Contribution

A simplified method for detecting mRNA isoforms using single-gene/transcript mapping is proposed and validated.

## Key findings

- Single-gene/transcript mapping can detect multiple Otx mRNA isoforms in sea urchin samples.
- RT-qPCR confirmed the presence of Otx mRNA isoforms in related species and developmental stages.
- The method works well for genes without overlapping exons and introns, like Otx in sea urchins.

## Abstract

Determination of mRNA isoform expression is important for studying tissue-specific gene regulation and the generation of protein diversity. We tested the potential of mapping to a single-gene/transcript for the qualitative examination of mRNA isoform expression produced from the same gene, including cases where several mRNA species are expressed in samples. Using single-gene/transcript mapping, we analyzed expression of the Otx mRNA isoforms in some mRNA libraries of the sea urchin Strongylocentrotus purpuratus available in GenBank. The presence of expressed Otx mRNA isoforms was verified by RT-qPCR in the same tissues and developmental stages of the closely related species Strongylocentrotus intermedius. We found that single-gene/transcript mapping can be easily applied to genes with a simple structure lacking overlapping exons and introns, such as Otx genes in sea urchins.

The identification of mRNA isoforms in biological samples is crucial for studying tissue- and cell-specific isoform expression, activity of tissue-specific promoters, alternative splicing events, and alternative polyadenylation signals in genes. For single or several genes, expressed mRNA isoforms can be found using RT-PCR and RT-qPCR. Available transcriptome short-read archives deposited in GenBank or as laboratory data can be used to identify mRNA isoforms instead of or prior to wet analysis by other methods in eukaryotic organisms with annotated genomes. However, isoform expression analysis requires advanced bioinformatics skills and may be time-consuming. In addition, this analysis generates a large amount of unnecessary data. To detect mRNA isoforms encoded by one gene of interest, screening of expressed mRNAs in NGS data can be simplified by mapping NGS short reads to a single-gene or transcript sequence. Using single-gene/transcript mapping, we analyzed the expression of the Otx gene at the mRNA isoform level in some embryonic and adult tissue mRNA libraries of the sea urchin Strongylocentrotus purpuratus available in GenBank. The presence of expressed Otx mRNA isoforms was confirmed by RT-qPCR in the same tissues and at the same developmental stages of the closely related species Strongylocentrotus intermedius. We showed that single-gene/transcript mapping is a suitable approach for qualitative evaluation of the expression of mRNA isoforms and recognition of at least two expressed isoforms in the same biological sample.