Long-Read: Nanopore Full-Length Transcriptome Sequencing
Posted by kiko on November 29th, 2023
Nanopore full-length transcriptome sequencing is an innovative technique that utilizes Oxford Nanopore Technologies' (ONT) sequencing platform to obtain comprehensive and uninterrupted high-quality full-length sequences of transcripts. This cutting-edge approach enables researchers to accurately identify various structural variations within transcripts, including alternative splicing, gene fusion, selective polyadenylation of alternative polyadenylation sites (APAs), allele-specific expression, and other alterations in transcript structure. Additionally, this method facilitates precise quantification of transcript expression levels, encompassing both messenger RNA (mRNA) and polyA+ long non-coding RNA (lncRNA).
Nanopore Sequencing vs. Next-Generation Transcriptome Sequencing
The key differences between Nanopore full-length transcriptome sequencing and next-generation transcriptome sequencing platforms are primarily related to their sequencing technology, read length, sample processing, and data output.
Nanopore Sequencing: Enhancing Transcript Expression Quantification
Nanopore full-length transcriptome sequencing presents a myriad of advantages that significantly contribute to its heightened accuracy in quantifying transcript expression levels compared to second-generation transcriptome sequencing methodologies. The following salient factors underscore the superior accuracy of Nanopore sequencing in transcript expression quantification:
Nanopore Sequencing: Detection of Structural Variants
Nanopore-based full-length transcriptome sequencing surpasses second-generation methods in the precise identification of structural variants, such as alternative splicing (AS), selective polyadenylation (APA), gene fusion, and complex transcripts, due to its distinct array of advantages:
Nanopore Sequencing: GC Content and Length Preference
Nanopore full-length transcriptome sequencing showcases distinct proclivities concerning GC content and transcript length preference, in contrast to NGS transcriptome sequencing. These variances carry profound ramifications for the precision and fidelity of gene expression quantification and structural variation analysis. Allow us to explore these disparities in greater depth:
a) Nanopore Full-Length Transcriptome Sequencing: Harnessing the long-read capability, Nanopore sequencing exhibits a remarkable capacity to provide a more equitable representation of GC content across transcripts. Notably, the long-read datasets derived from Nanopore technology demonstrate a discernible reduction in GC preference as compared to their short-read counterparts. This advantageous attribute alleviates potential biases arising from fluctuations in GC content, thereby facilitating more accurate and robust quantification of gene expression levels. Such improvements are particularly pronounced when assessing regions with diverse GC-rich or GC-poor compositions.
b) Next-Generation Transcriptome Sequencing: In contrast, short-read sequencing technologies, typified by conventional next-generation cDNA sequencing, have shown a propensity for GC content preference. This proclivity can induce distortions in the quantification of gene expression, especially in genomic loci characterized by extreme GC content. The consequent biases may impede the accurate identification of differentially expressed genes and transcript isoforms, undermining the fidelity of the analysis.
a) Nanopore Full-Length Transcriptome Sequencing: Capitalizing on its long-read capabilities, Nanopore sequencing generates datasets spanning the entirety of transcripts. As a result, the observed length bias in nanopore sequencing data is significantly ameliorated when compared to short-read datasets. This represents a substantial advantage, ensuring a comprehensive and representative coverage of transcripts with varying lengths. The technology thereby enables precise quantification of both short and long transcripts, enhancing the overall fidelity of the transcriptome analysis.
b) NGS: Short-read sequencing technologies inherently contend with a length preference bias. The necessity for transcript fragmentation to fit within limited short read lengths elevates the risk of overlooking extended transcripts or segments with substantial length. Consequently, this length bias can lead to incomplete characterization of transcript isoforms, particularly those featuring lengthy exons or intricate exon-exon junctions, thereby potentially compromising the accuracy of the analysis.
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About the Authorkiko
Joined: November 27th, 2018
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