Targeted, High-Resolution Rna Sequencing of Non-Coding Genomic Regions Associated With Neuropsychiatric Functions

Abstract

The human brain is one of the last frontiers of biomedical research. Genome-wide association studies (GWAS) have succeeded in identifying thousands of haplotype blocks associated with a range of neuropsychiatric traits, including disorders such as schizophrenia, Alzheimer's and Parkinson's disease. However, the majority of single nucleotide polymorphisms (SNPs) that mark these haplotype blocks fall within noncoding regions of the genome, hindering their functional validation. While some of these GWAS loci may contain cis-acting regulatory DNA elements such as enhancers, we hypothesized that many are also transcribed into non-coding RNAs that are missing from publicly available transcriptome annotations. Here, we use targeted RNA capture ('RNA CaptureSeq') in combination with nanopore long-read cDNA sequencing to transcriptionally profile 1,023 haplotype blocks across the genome containing non-coding GWAS SNPs associated with neuropsychiatric traits, using post-mortem human brain tissue from three neurologically healthy donors. We find that the majority (62%) of targeted haplotype blocks, including 13% of intergenic blocks, are transcribed into novel, multi-exonic RNAs, most of which are not yet recorded in GENCODE annotations. We validated our findings with short-read RNAseq, providing orthogonal confirmation of novel splice junctions and enabling a quantitative assessment of the long-read assemblies. Many novel transcripts are supported by independent evidence of transcription including cap analysis of gene expression (CAGE) data and epigenetic marks, and some show signs of potential functional roles. We present these transcriptomes as a preliminary atlas of noncoding transcription in human brain that can be used to connect neurological phenotypes with gene expression.2

Functional annotation of human long noncoding RNAs via molecular phenotyping

“…Molecular phenotyping is a powerful and generally more sensitive to knockdown-mediated changes platform to reveal the functional relevance of lncRNAs that cannot be observed based on the cellular phenotypes alone. With additional molecular profiling techniques, such as RNA duplex maps in living cells to decode common structural motifs , and Oxford Nanopore Technology (ONT) to annotate the full-length variant isoforms of lncRNAs (Hardwick et al 2019), the structure-to-functional relationship of lncRNAs may be elucidated further in the future.…”

Improving the diversity of captured full-length isoforms using a normalized single-molecule RNA-sequencing method

“…There are also other strategies to enrich rare transcripts and improve the diversity of transcripts. For example, targeted sequencing has been widely adopted to couple with long-read RNA sequencing, and disclosed a large number of important transcripts that were out of detection by traditional SMS methods 8 , 31 – 33 . Compared with cDNA normalization, targeted sequencing showed stronger capability in improving the diversity of sequenced isoforms.…”

“…First, cDNA normalization can be performed to any organism while targeted sequencing can only be applied to model organisms with genome sequenced. Second, selection of interesting genome regions is a prerequisite for targeted RNA sequencing 8 , 32 , 33 . cDNA normalization does not have such limitations, and therefore is suitable for more general transcriptome studies.…”

Mechanisms of Human intelligence — From RNA and Synapse to Broadband

“…Significantly though, about 40% (of "Long" ncRNA alone) are found only in the brain. 26,7 The conclusion here is that much of this remainder probably serves as modifiable basic action codes, either for random (Darwinian) action-trials, or as important inherited behavioural traits, (mannerisms which are thereby easily explained! ).…”