mRNA sequencing (mRNA-Seq) evaluates the transcriptome of disease states and biological processes. mRNA-Seq is a precise method for measuring gene expression. Unlike small reads typically produced by next-generation transcriptome sequencing, long reads help detect full-length transcripts, gene fusions, and allele-specific expression. In this blog post, we describe several advantages of long-read over short-read sequencing when used for transcriptome analysis.
PacBio's SMRT sequencing uses DNA polymerase to perform uninterrupted template-directed synthesis. It is free from the PCR amplification step and, as a result, removes amplification bias and provides uniform coverage across the transcriptome. It produces extraordinarily long reads with average lengths of 4200 to 8500 bp, which enhances the detection of novel transcript structures.
The transcriptome of species has a high degree of complexity. It contains multiple types of coding and noncoding RNAs. Long-read technologies such as Pacific Biosciences (PacBio) single-molecule real-time (SMRT) sequencing and Oxford Nanopore Technologies (ONT) nanopore sequencing are improving accuracy and throughput compared to short-reads. Long reads provide an accurate, high-resolution view of transcriptomes and isoform identification. Additionally, it accurately quantifies overlapping transcripts and increases the percentage of alignable reads. In addition to providing gene expression quantification, long-read sequencing provides full-length transcript resolution and enables the identification of unknown genes and alternatively spliced transcripts.
Compared to short reads, long reads produced by third-generation sequencers tend to overlap better with other reads. As a result, re-assembling the RNA pieces in their proper sequence is straightforward. Additionally, long-reads are more likely to cover the repetitive region, enabling the construction of whole transcript assemblies with fewer gaps.
Briefly, although short-read sequencing and long-read sequencing perform equally for quantification of gene expression, the latter is efficient in identifying transcript isoforms and provides better transcript resolution with a higher mean contig length. Higher contig length enables full-length de Novo assembly of the transcriptome.
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