![]() ![]() There is a growing need for public health and veterinary laboratories to perform whole genome sequencing (WGS) for monitoring antimicrobial resistance (AMR) and protecting the safety of people and animals. To understand the contribution of variants to transcript expression regulation, long-read transcriptome data are generated from the GTEx resource, and a new software package to perform allele-specific analysis is developed. Finally, we used this dataset to enhance variant interpretation and study rare variants leading to aberrant splicing patterns. ![]() We were able to perturb the transcript structure upon knockdown of PTBP1, an RNA binding protein that mediates splicing, thereby finding genetic regulatory effects that are modified by the cellular environment. We characterized allele-specific expression and transcript structure events, providing new insights into the specific transcript alterations caused by common and rare genetic variants and highlighting the resolution gained from long-read data. We developed a new computational package, LORALS, to analyse the genetic effects of rare and common variants on the transcriptome by allele-specific analysis of long reads. We identified just over 70,000 novel transcripts for annotated genes, and validated the protein expression of 10% of novel transcripts. In this Article, we present a large human long-read RNA-seq dataset using the Oxford Nanopore Technologies platform from 88 samples from Genotype-Tissue Expression (GTEx) tissues and cell lines, complementing the GTEx resource. The advent of long-read sequencing technologies offers the opportunity to study the role of genetic variation in transcript structure8–16. Regulation of transcript structure generates transcript diversity and plays an important role in human disease1–7. We also explore the use of this approach for rapid evaluation of sequencing library metrics by implementing a real-time analysis workflow. We highlight this capability by producing and benchmarking RNA-seq, ChIP-seq, as well as regular and target-enriched Tn5 libraries. This longer DNA is ideally suited for the ONT MinION, and after sequencing, the tandem repeats in the resulting raw reads can be converted into high-accuracy consensus reads with similar error rates to that of the Illumina MiSeq. This results in longer DNA molecules containing tandem repeats of the original short library molecules. Here we show that we can make short-read Illumina libraries compatible with the ONT MinION by using the R2C2 method to circularize and amplify the short library molecules. Among a new generation of sequencing technologies, Oxford Nanopore Technologies (ONT) holds a unique position because the ONT MinION, an error-prone long-read sequencer, is associated with little to no capital cost. Although other short-read sequencing technologies exist, the ubiquity of Illumina sequencers in sequencing core facilities, and the high capital costs of these technologies have limited their adoption. Hundreds of different assays exist today to take advantage of Illumina short-read sequencers, the predominant short-read sequencing technology available today. High-throughput short-read sequencing has taken on a central role in research and diagnostics. ![]()
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