Long-read transcriptome sequencing reveals abundant promoter diversity in distinct molecular subtypes of gastric cancer

Abstract

Background Deregulated gene expression is a hallmark of cancer; however, most studies to date have analyzed short-read RNA sequencing data with inherent limitations. Here, we combine PacBio long-read isoform sequencing (Iso-Seq) and Illumina paired-end short-read RNA sequencing to comprehensively survey the transcriptome of gastric cancer (GC), a leading cause of global cancer mortality. Results We performed full-length transcriptome analysis across 10 GC cell lines covering four major GC molecular subtypes (chromosomal unstable, Epstein-Barr positive, genome stable and microsatellite unstable). We identify 60,239 non-redundant full-length transcripts, of which > 66% are novel compared to current transcriptome databases. Novel isoforms are more likely to be cell line and subtype specific, expressed at lower levels with larger number of exons, with longer isoform/coding sequence lengths. Most novel isoforms utilize an alternate first exon, and compared to other alternative splicing categories, are expressed at higher levels and exhibit higher variability. Collectively, we observe alternate promoter usage in 25% of detected genes, with the majority (84.2%) of known/novel promoter pairs exhibiting potential changes in their coding sequences. Mapping these alternate promoters to TCGA GC samples, we identify several cancer-associated isoforms, including novel variants of oncogenes. Tumor-specific transcript isoforms tend to alter protein coding sequences to a larger extent than other isoforms. Analysis of outcome data suggests that novel isoforms may impart additional prognostic information. Conclusions Our results provide a rich resource of full-length transcriptome data for deeper studies of GC and other gastrointestinal malignancies.

Isoform Age - Splice Isoform Profiling Using Long-Read Technologies

“…A recent study of ten gastric cancer cell lines using PacBio's Iso-Seq technology identified approximately 39,000 novel isoforms, including for the known oncogenes ERBB2 and CD44. Alternative promoters were frequently used in gastric cancer cells which often resulted in altered downstream CDS and 3′ UTRs (Huang et al, 2021). Full-length transcripts, using both ONT and PacBio reads, have also been sequenced to identify variants involved in treatment resistance in triple negative breast and lung cancer cell lines (Lian et al, 2019;Seki et al, 2019;Oka et al, 2021).…”

A Genome-Wide Investigation of Effects of Aberrant DNA Methylation on the Usage of Alternative Promoters in Hepatocellular Carcinoma

“…The abnormally upregulated promoter activities in HCC may lead to the changes of the CDS region and produce new protein subtypes, as reported (12). For example, the upregulated prmtr.14927 in MET may lead to the accumulation of a 960-aa protein isoform that lacks the SEMA domain in HCC (Supplementary Figure 2B).…”

Single-Cell Proteomic Analysis Dissects the Complexity of Tumor Microenvironment in Muscle Invasive Bladder Cancer

“…Subgrouping of patients based on molecular characteristics has considerably affected the selection of therapeutic regimens and the forecasting of therapeutic responses and prognoses [44]. The establishment of tumor molecular subtypes has advanced our understanding of tumor genotypes and phenotypes, presenting a potential application in therapeutics and prognosis estimation [61,62]. At present, the ConMC divides MIBC into six molecular subtypes and systematically exposes specific differentiation patterns, mutation genes, histology, and overall survival rates in the six molecular subtypes [45].…”