In this webinar, Richard Kuo of the Roslin Institute discusses how his team addressed RNA degradation by using Lexogen’s cap-specific TeloPrime method to prepare full-length cDNA libraries. To reduce overabundant genes, Kuo and colleagues experimented with two methods of cDNA normalization.
He presents his team’s results using these novel methods of cDNA preparation for Iso-Seq sequencing and also covers some basics of LRTseq and analysis pipelines for Iso-Seq (from raw data to transcriptome annotation).
Abstract
This webinar discusses novel long-read transcript sequencing (LRTseq) methods for transcriptome annotation that could increase the efficiency and accuracy of future sequencing projects.
LRTseq technologies, such as Pacific Biosciences’ Iso-Seq and Oxford Nanopore’s cDNA sequencing, have the power to provide rapid high-quality de novo transcriptome annotations. However, standard cDNA library preparation methods for LRTseq capture a significant amount of degraded RNA and are often overpopulated with highly expressed genes.
Degraded RNA reduces the efficiency of sequencing and introduces uncertainty with respect to predicted transcription start sites. Highly expressed genes can dominate LRTseq data, resulting in a loss of coverage for lower expressed genes. This typically results in missing genes and alternative transcripts in the final genome annotation.
Speaker’s biography
Richard Kuo is a Bioinformatician and PhD Student at the Roslin Institute of the University of Edinburgh specializing in transcriptomics and lncRNA. He is the creator and developer of TAMA, a software package for downstream Iso-Seq data analyses. He is also involved with the Functional Annotation of Animal Genomes (FAANG) consortium and the Vertebrate Genome Project.
