Depletion of hemoglobin transcripts and long-read sequencing improves the transcriptome annotation of the polar bear (Ursus maritimus)Publication
Date: 19th July 2019 | Source: Frontiers in Genetics
Transcriptome studies evaluating whole blood and tissues are often confounded by overrepresentation of highly abundant transcripts. These abundant transcripts are problematic as they compete with and prevent the detection of rare RNA transcripts, obscuring their biological importance. This issue is more pronounced when using long-read sequencing technologies for isoform-level transcriptome analysis, as they have relatively lower throughput compared to short-read sequencers. As a result long-read based transcriptome analysis is prohibitively expensive for non-model organisms. While there are off-the-shelf kits available for select model organisms capable of depleting highly abundant transcripts for alpha (HBA) and beta (HBB) hemoglobin, they are unsuitable for non-model organisms. To address this, we have adapted the recent CRISPR/Cas9 based depletion method (Depletion of Abundant Sequences by Hybridization) for long-read full-length cDNA sequencing approaches that we call Long-DASH. Using a recombinant Cas9 protein with appropriate guide RNAs, full-length hemoglobin transcripts can be depleted in vitro prior to performing any short- and long-read sequencing library preparations. Using this method, we sequenced depleted full-length cDNA in parallel using both our Oxford Nanopore Technology (ONT) based R2C2 long-read approach, as well as the Illumina short-read based Smart-seq2 approach. To showcase this, we have applied our methods to create an isoform-level transcriptome from whole blood samples derived from three polar bears (Ursus maritimus). Using Long-DASH, we succeeded in depleting hemoglobin transcripts and generated deep Smart-seq2 Illumina datasets and 3.8 million R2C2 full-length cDNA consensus reads. Applying Long-DASH with our isoform identification pipeline, Mandalorion we discovered ~6,000 high-confidence isoforms and a number of novel genes. This indicates there is a high diversity of gene isoforms within Ursus maritimus not yet reported. This reproducible and straightforward approach has not only improved the polar bear transcriptome annotations but will serve as the foundation for future efforts to investigate transcriptional dynamics within the 19 polar bear subpopulations around the Arctic.
‘To provide an accurate isoform-level transcriptome annotation for non-model organisms, long-read sequencing technology is required to sequence full-length cDNA molecules’.
Byrne et al