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Comparison of single-nuclei 5’ versus 3’-RNA-seq approaches: utility for somatic variant detection

ABSTRACT: Somatic mosaic variants are a major cause of human disease, including cancer and focal epilepsies, but can be challenging to study due to their mosaicism in bulk tissue biopsies. Coupling single-cell genotype and transcriptomic data has potential to provide insight into the role somatic variants play in disease etiology, such as by determining what cell types are affected or how the mutations affect gene expression. Here, we asked whether commonly used single-cell 3’- or 5’-RNA-sequencing assays can be used to derive single-cell genotype data for a priori known variants that are located near to either end of a transcript. To that end, we compared performance of commercially available single-cell 3’- and 5’- gene expression kits using resected brain samples from three pediatric patients with focal epilepsy. We quantified the ability to detect genetic variants in single-cell datasets depending on distance from the transcript end. Finally, we demonstrated the ability to identify affected cell types in a patient with a RHEB somatic variant causing an epilepsy-associated cortical malformation. Our results demonstrate that native single-cell 3’ or 5’-RNA-sequencing data can be used successfully to genotype single-cells for somatic variants that are expressed within proximity to a transcript end.

ORGANISM(S): Homo sapiens

PROVIDER: GSE210670 | GEO | 2023/01/09

REPOSITORIES: GEO

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Project description:The epilepsies represent one of the most common neurological disorders. Mesial temporal lobe epilepsies (MTLE) are the most frequent form of partial epilepsies and display frequent resistance to anti-epileptic drugs thus representing a major health care problem. In TLE, the origin of seizure activity typically involves the hippocampal formation, which displays major neuropathological features, described with the term hippocampal sclerosis (HS). HS is the most frequent pathological substrate of refractory mesial temporal lobe epilepsy. Complex partial seizures (CPS) are the predominant seizure type associated with medial temporal lobe epilepsy. MTLE is commonly due to mesial temporal sclerosis (MTS). The biology underlying the epilepstic seizures and the transcriptome associated to the seizure in intractable medial temporal lobe epilepsy is ill understood. The aim of the study was to identify potential biomarkers that could identify epileptic seizure. Thus we performed transcriptome profiling of ten medial temporal lobe epilepsy cases which are resistant to the drug and underwent temporal lobectomy. The cases constitutes of patients with intractable complex partial seizure, treated medically and have undergone detailed presurgical evaluation and subjected to surgery for standard temporal lobectomy and amygdalo-hippocampectomy. The spiking areas identified after the electrocorticography will form the test tissues, which compared with the nonspiking areas removed during the surgery, from the same patient. This could probably form one of the appropriate controls, as test and control are from same patient, which eliminates the genome variations that could incur due to the comparison with the tissues from the another patient. Also this could get rid of expression changes due to the treatments undergone by the patient. We performed two color microarray wherein we labled seizure focus (spiking area) with Cy5 and non-seizure region tissues (non-spiking) with Cy3. As a strategy to test the possibility of potential diagnostic biomarkers we are intended to test the differentially regulated molecules in an independent set of epilepsy samples. Two color experiment

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Comparison of single-nuclei 5’ versus 3’-RNA-seq approaches: utility for somatic variant detection

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