RNA isoform expression landscape of the human dorsal root ganglion generated from long-read sequencing.

ABSTRACT: Splicing is a posttranscriptional RNA processing mechanism that enhances genomic complexity by creating multiple isoforms from the same gene. We aimed to characterize the isoforms expressed in the human peripheral nervous system, with the goal of creating a resource to identify novel isoforms of functionally relevant genes associated with somatosensation and nociception. We used long-read sequencing to document isoform expression in the human dorsal root ganglia from 3 organ donors and validated in silico by confirming expression in short-read sequencing from 3 independent organ donors. Nineteen thousand five hundred forty-seven isoforms of protein-coding genes were detected and validated. We identified 763 isoforms with at least one previously undescribed splice junction. Previously unannotated isoforms of multiple pain-associated genes, including ASIC3 , MRGPRX1 , and HNRNPK , were identified. In the novel isoforms of ASIC3 , a region comprising approximately 35% of the 5'UTR was excised. By contrast, a novel splice junction was used in isoforms of MRGPRX1 to include an additional exon upstream of the start codon, consequently adding a region to the 5'UTR. Novel isoforms of HNRNPK were identified, which used previously unannotated splice sites to both excise exon 14 and include a sequence in the 3' end of exon 13. This novel insertion is predicted to introduce a tyrosine phosphorylation site potentially phosphorylated by SRC. We also independently confirm a recently reported DRG-specific splicing event in WNK1 that gives insight into how painless peripheral neuropathy occurs when this gene is mutated. Our findings give a clear overview of mRNA isoform diversity in the human dorsal root ganglia obtained using long-read sequencing.

RNA isoform expression landscape of the human dorsal root ganglion (DRG) generated from long read sequencing.

Splicing is a post-transcriptional RNA processing mechanism that enhances genomic complexity by creating multiple isoforms from the same gene. Diversity in splicing in the mammalian nervous system is associated with neuronal development, synaptic function and plasticity, and is also associated with diseases of the nervous system ranging from neurodegeneration to chronic pain. We aimed to characterize the isoforms expressed in the human peripheral nervous system, with the goal of creating a resource to identify novel isoforms of functionally relevant genes associated with somatosensation and nociception. We used long read sequencing (LRS) to document isoform expression in the human dorsal root ganglia (hDRG) from 3 organ donors. Isoforms were validated in silico by confirming expression in hDRG short read sequencing (SRS) data from 3 independent organ donors. 19,547 isoforms of protein-coding genes were detected using LRS and validated with SRS and strict expression cutoffs. We identified 763 isoforms with at least one previously undescribed splice-junction. Previously unannotated isoforms of multiple pain-associated genes, including ASIC3, MRGPRX1 and HNRNPK were identified. In the novel isoforms of ASIC3, a region comprising ~35% of the 5'UTR was excised. In contrast, a novel splice-junction was utilized in isoforms of MRGPRX1 to include an additional exon upstream of the start-codon, consequently adding a region to the 5'UTR. Novel isoforms of HNRNPK were identified which utilized previously unannotated splice-sites to both excise exon 14 and include a sequence in the 5' end of exon 13. The insertion and deletion in the coding region was predicted to excise a serine-phosphorylation site favored by cdc2, and replace it with a tyrosine-phosphorylation site potentially phosphorylated by SRC. We also independently confirm a recently reported DRG-specific splicing event in WNK1 that gives insight into how painless peripheral neuropathy occurs when this gene is mutated. Our findings give a clear overview of mRNA isoform diversity in the hDRG obtained using LRS. Using this work as a foundation, an important next step will be to use LRS on hDRG tissues recovered from people with a history of chronic pain. This should enable identification of new drug targets and a better understanding of chronic pain that may involve aberrant splicing events.

Performance evaluation of a non-invasive one-step multiplex RT-qPCR assay for detection of SARS-CoV-2 direct from saliva.

Polymerase chain reaction (PCR) has proven to be the gold-standard for SARS-CoV-2 detection in clinical settings. The most common approaches rely on nasopharyngeal specimens obtained from swabs, followed by RNA extraction, reverse transcription and quantitative PCR. Although swab-based PCR is sensitive, swabbing is invasive and unpleasant to administer, reducing patient compliance for regular testing and resulting in an increased risk of improper sampling. To overcome these obstacles, we developed a non-invasive one-step RT-qPCR assay performed directly on saliva specimens. The University of Nottingham Asymptomatic Testing Service protocol simplifies sample collection and bypasses the need for RNA extraction, or additives, thus helping to encourage more regular testing and reducing processing time and costs. We have evaluated the assay against the performance criteria specified by the UK regulatory bodies and attained accreditation (BS EN ISO/IEC 17,025:2017) for SARS-CoV-2 diagnostic testing by the United Kingdom Accreditation Service. We observed a sensitivity of 1 viral copy per microlitre of saliva, and demonstrated a concordance of > 99.4% between our results and those of other accredited testing facilities. We concluded that saliva is a stable medium that allows for a highly precise, repeatable, and robust testing method.

Direct RT-qPCR Assay for the Detection of SARS-CoV-2 in Saliva Samples.

Since mid-2020 there have been complexities and difficulties in the standardisation and administration of nasopharyngeal swabs. Coupled with the variable and/or poor accuracy of lateral flow devices, this has led to increased societal 'testing fatigue' and reduced confidence in test results. Consequently, asymptomatic individuals have developed reluctance towards repeat testing, which remains the best way to monitor COVID-19 cases in the wider population. On the other hand, saliva-based PCR, a non-invasive, highly sensitive, and accurate test suitable for everyone, is gaining momentum as a straightforward and reliable means of detecting SARS-CoV-2 in symptomatic and asymptomatic individuals. Here, we provide an itemised list of the equipment and reagents involved in the process of sample submission, inactivation and analysis, as well as a detailed description of how each of these steps is performed.