Tonight I watched the Nanopore Community Meeting 2022 session by Alisa MacCalman from the University of Exeter in the UK. The title of the session was “Ultra-deep targeted transcript sequencing identifies isoform diversity across human pancreatic development.” MacCalman spoke about pancreatic development and that most of the knowledge is from mouse models. They are interested in genomic regulation and more recently in transcriptome and alternative splicing. Alternative splicing is when multiple different mRNA molecules originate from a gene. MacCalman noted that isform diversity is developmentally dynamic and increases the complexity of gene expression. To look at RNA splicing, RNA-seq is needed. However, short reads are limited. ONT cDNA sequencing can identify exons. MacCalman’s group has performed isoform analysis in the brain. MacCalman has a unique cohort of 122 samples and selected 31 human fetal pancreas samples spanning 6-21 weeks post conception. These samples have also been subjected to epigenomic analysis. These samples were selected by RNA RIN values (above 8). RNA was extracted, reverse transcription and PCR synthesis performed, and sequencing conducted on the PromethION. The output was huge: 150 Gbases. Novel isoforms were found… very frequently. With Nanopore sequencing, alternative splicing events were studied, focusing on their distribution. MacCalman is studying differential transcript expression between the pancreas and cortex. They have developed a system and have access to the samples to continue discovering and learning about isoform diversity!
