Transcript Profiling Using Long-Read Sequencing Technologies

Methods in Molecular Biology
Anthony BayegaJiannis Ragoussis

Abstract

RNA sequencing using next-generation sequencing (NGS, RNA-Seq) technologies is currently the standard approach for gene expression profiling, particularly for large-scale high-throughput studies. NGS technologies comprise short-read RNA-Seq (dominated by Illumina) and long-read RNA-Seq technologies provided by Pacific Bioscience (PacBio) and Oxford Nanopore Technologies (ONT). Although short-read sequencing technologies are the most widely used, long-read technologies are increasingly becoming the standard approach for de novo transcriptome assembly and isoform expression quantification due to the complex nature of the transcriptome which consists of variable lengths of transcripts and multiple alternatively spliced isoforms for most genes. In this chapter, we describe experimental procedures for library preparation, sequencing, and associated data analysis approaches for PacBio and ONT with a major focus on full length cDNA synthesis, de novo transcriptome assembly, and isoform quantification.

Citations

Sep 21, 2019·Human Genetics·Mohit K MidhaKuo-Ping Chiu
Aug 1, 2020·Frontiers in Genetics·Spyros OikonomopoulosJiannis Ragoussis
Jan 6, 2021·Nature Communications·Kristoffer Sahlin, Paul Medvedev
Mar 7, 2021·International Journal of Molecular Sciences·Izarbe Aísa-MarínGemma Marfany

Related Concepts

cDNA Library
Nested Transcripts
Sequence Determinations, RNA
Computational Molecular Biology
Protein Isoforms
MRNA Differential Display
High-Throughput Nucleotide Sequencing
Gene Expression Profiles
DNA, Complementary
Gene Expression

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