Methodologies for Transcript Profiling Using Long-Read Technologies

Abstract

RNA sequencing using next-generation sequencing technologies (NGS) is currently the standard approach for gene expression profiling, particularly for large-scale highthroughput studies. NGS technologies comprise high throughput, cost efficient shortread RNA-Seq, while emerging single molecule, long-read RNA-Seq technologies have enabled new approaches to study the transcriptome and its function. The emerging single molecule, long-read technologies are currently commercially available by Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT), while new methodologies based on short-read sequencing approaches are also being developed in order to provide long range single molecule level information-for example, the ones represented by the 10x Genomics linked read methodology. The shift toward longread sequencing technologies for transcriptome characterization is based on current increases in throughput and decreases in cost, making these attractive for de novo transcriptome assembly, isoform expression quantification, and in-depth RNA species analysis. These types of analyses were challenging with standard short sequencing approaches, due to the complex nature of the transcriptome, which consists of variable lengths of transcripts and multiple alternatively spliced isoforms for most genes, as well as the high sequence similarity of highly abundant species of RNA, such as rRNAs. Here we aim to focus on single molecule level sequencing technologies and single-cell technologies that, combined with perturbation tools, allow the analysis of complete RNA species, whether short or long, at high resolution. In parallel, these tools have opened new ways in understanding gene functions at the tissue, network, and pathway levels, as well as their detailed functional characterization. Analysis of the epi-transcriptome, including RNA methylation and modification and the effects of such modifications on biological systems is now enabled through direct RNA sequencing instead of classical indirect approaches. However, many difficulties and challenges remain, such as methodologies to generate full-length RNA or cDNA libraries from all different species of RNAs, not only poly-A containing transcripts, and the identification of allele-specific transcripts due to current error rates of single molecule technologies, while the bioinformatics analysis on long-read data for accurate identification of 5 and 3 UTRs is still in development.

The impact of biological sex on alternative splicing

“…Our study provides a comprehensive atlas of genes showing sex-biased AS across a range of tissues. AS is becoming more easily accessible thanks to long-read transcriptome sequencing ( 56 ), and we anticipate that more detailed investigations of sex differences in specific diseases will be valuable for advancing precision medicine for males and females.…”

Systematic assessment of long-read RNA-seq methods for transcript identification and quantification

“…There is a growing trend of using long-read RNA-seq (lrRNA-seq) data for transcript identification and quantification, primarily with Oxford Nanopore Technologies (ONT) and Pacific Biosciences (PacBio) platforms [1][2][3][4] . Consequently, there is a need to evaluate these approaches for transcriptome analysis to compare the impact of different sequencing platforms, multiple sequencing library preparation methods, and computational analysis methods (Reviewed in [5][6][7][8] ).…”

A de novo Full-Length mRNA Transcriptome Generated From Hybrid-Corrected PacBio Long-Reads Improves the Transcript Annotation and Identifies Thousands of Novel Splice Variants in Atlantic Salmon

“…Full-length transcriptomes have a range of useful applications ( Oikonomopoulos et al, 2020 ). Among those mentioned, we propose that our high-quality full-length transcriptomes may serve as references in expression analyses.…”