Poly(A) capture full length cDNA sequencing improves the accuracy and detection ability of transcript quantification...

Area of Science:

• Molecular Biology
• Genomics
• Bioinformatics

Background:

• Full-length double-strand cDNA sequencing is a valuable RNA-Seq method for analyzing gene transcription levels and alternative splicing variants.
• This method offers advantages such as library creation from small samples and compatibility with both short-read and long-read sequencers.
• Previous studies identified non-specific genomic DNA amplification as a significant issue affecting transcriptome analysis accuracy.

Purpose of the Study:

• To confirm and address the issue of non-specific genomic DNA amplification in full-length double-strand cDNA sequencing.
• To evaluate two methods, DNase I-treated and poly(A) capture, for mitigating genomic DNA contamination.
• To assess the impact of these methods on transcriptome analysis, including gene expression quantification and alternative splicing detection.

Main Methods:

• Investigated RNA-Seq library preparation from genomic DNA to confirm non-specific amplification potential.
• Compared DNase I-treated full-length double-strand cDNA sequencing with standard methods.
• Evaluated poly(A) capture full-length double-strand cDNA sequencing for its efficacy in reducing genomic DNA contamination.

Main Results:

• Confirmed that RNA-Seq libraries can be produced from genomic DNA, leading to non-specific amplification.
• DNase I treatment unexpectedly increased genomic DNA amplification and reduced detected expressing genes.
• Poly(A) capture significantly reduced non-specific genomic DNA amplification, enhancing accuracy, gene detection, and splicing event identification.

Conclusions:

• Poly(A) capture full-length double-strand cDNA sequencing is a superior method for improving transcript quantification accuracy.
• This method enhances the detection of alternative splicing events and their expression patterns.
• The findings are expected to aid in determining the significance of DNA variants in splicing events.