Long-read cDNA sequencing reveals novel isoforms and spliceosome-mutant-enriched transcripts in AML and MDS

Area of Science:

• Genomics
• Molecular Biology
• Cancer Research

Background:

• Alternative splicing in cancer transcriptomes is poorly understood due to limitations of short-read sequencing.
• Resolving complete transcript structures is crucial for characterizing cancer's molecular landscape.

Purpose of the Study:

• To characterize the alternative splicing landscape in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) using long-read sequencing.
• To identify novel RNA isoforms and their functional relevance in spliceosome-mutant cancers.

Main Methods:

• Utilized Oxford Nanopore cDNA sequencing to generate nearly 2 billion long reads from 71 human samples (48 AML/MDS, 23 healthy controls).
• Analyzed samples, including those with splicing-factor gene mutations (SRSF2, U2AF1, SF3B1).
• Performed proteomic validation to confirm translation of novel isoforms and investigate nonsense-mediated decay.

Main Results:

• Identified 174,162 novel RNA isoforms not present in the reference transcriptome.
• Confirmed translation of many novel isoforms through proteomic analysis.
• Discovered isoforms enriched in spliceosome-mutant samples and evidence of nonsense-mediated decay regulation.

Conclusions:

• Long-read sequencing provides unprecedented resolution for alternative splicing analysis in cancer.
• The identified novel isoforms and their regulation offer new insights into AML and MDS pathogenesis.
• This dataset serves as a valuable resource for detecting new transcripts in existing short-read data.