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June 1, 2021  |  

A method for the identification of variants in Alzheimer’s disease candidate genes and transcripts using hybridization capture combined with long-read sequencing

Alzheimer’s disease (AD) is a devastating neurodegenerative disease that is genetically complex. Although great progress has been made in identifying fully penetrant mutations in genes such as APP, PSEN1 and PSEN2 that cause early-onset AD, these still represent a very small percentage of AD cases. Large-scale, genome-wide association studies (GWAS) have identified at least 20 additional genetic risk loci for the more common form of late-onset AD. However, the identified SNPs are typically not the actual risk variants, but are in linkage disequilibrium with the presumed causative variant (Van Cauwenberghe C, et al., The genetic landscape of Alzheimer disease: clinical implications and perspectives. Genet Med 2015;18:421-430). Long-read sequencing together with hybrid-capture targeting technologies provides a powerful combination to target candidate genes/transcripts of interest. Shearing the genomic DNA to ~5 kb fragments and then capturing with probes that span the whole gene(s) of interest can provide uniform coverage across the entire region, identifying variants and allowing for phasing into two haplotypes. Furthermore, capturing full-length cDNA from the same sample using the same capture probes can also provide an understanding of isoforms that are generated and allow them to be assigned to their corresponding haplotype. Here we present a method for capturing genomic DNA and cDNA from an AD sample using a panel of probes targeting approximately 20 late-onset AD candidate genes which includes CLU, ABCA7, CD33, TREM2, TOMM40, PSEN2, APH1 and BIN1. By combining xGen® Lockdown® probes with SMRT Sequencing, we provide completely sequenced candidate genes as well as their corresponding transcripts. In addition, we are also able to evaluate structural variants that due to their size, repetitive nature, or low sequence complexity have been un-sequenceable using short-read technologies.

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June 1, 2021  |  

A method for the identification of variants in Alzheimer’s disease candidate genes and transcripts using hybridization capture combined with long-read sequencing

Alzheimer’s disease (AD) is a devastating neurodegenerative disease that is genetically complex. Although great progress has been made in identifying fully penetrant mutations in genes such as APP, PSEN1 and PSEN2 that cause early-onset AD, these still represent a very small percentage of AD cases. Large-scale, genome-wide association studies (GWAS) have identified at least 20 additional genetic risk loci for the more common form of late-onset AD. However, the identified SNPs are typically not the actual causal variants, but are in linkage disequilibrium with the presumed causative variant (Van Cauwenberghe C, et al., The genetic landscape of Alzheimer disease: clinical implications and perspectives. Genet Med 2015;18:421-430).

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June 1, 2021  |  

Screening for causative structural variants in neurological disorders using long-read sequencing

Over the past decades neurological disorders have been extensively studied producing a large number of candidate genomic regions and candidate genes. The SNPs identified in these studies rarely represent the true disease-related functional variants. However, more recently a shift in focus from SNPs to larger structural variants has yielded breakthroughs in our understanding of neurological disorders.Here we have developed candidate gene screening methods that combine enrichment of long DNA fragments with long-read sequencing that is optimized for structural variation discovery. We have also developed a novel, amplification-free enrichment technique using the CRISPR/Cas9 system to target genomic regions.We sequenced gDNA and full-length cDNA extracted from the temporal lobe for two Alzheimer’s patients for 35 GWAS candidate genes. The multi-kilobase long reads allowed for phasing across the genes and detection of a broad range of genomic variants including SNPs to multi-kilobase insertions, deletions and inversions. In the full-length cDNA data we detected differential allelic isoform complexity, novel exons as well as transcript isoforms. By combining the gDNA data with full-length isoform characterization allows to build a more comprehensive view of the underlying biological disease mechanisms in Alzheimer’s disease. Using the novel PCR-free CRISPR-Cas9 enrichment method we screened several genes including the hexanucleotide repeat expansion C9ORF72 that is associated with 40% of familiar ALS cases. This method excludes any PCR bias or errors from an otherwise hard to amplify region as well as preserves the basemodication in a single molecule fashion which allows you to capture mosaicism present in the sample.

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April 21, 2020  |  

The landscape of SNCA transcripts across synucleinopathies: New insights from long reads sequencing analysis

Dysregulation of alpha-synuclein expression has been implicated in the pathogenesis of synucleinopathies, in particular Parkinsontextquoterights Disease (PD) and Dementia with Lewy bodies (DLB). Previous studies have shown that the alternatively spliced isoforms of the SNCA gene are differentially expressed in different parts of the brain for PD and DLB patients. Similarly, SNCA isoforms with skipped exons can have a functional impact on the protein domains. The large intronic region of the SNCA gene was also shown to harbor structural variants that affect transcriptional levels. Here we apply the first study of using long read sequencing with targeted capture of both the gDNA and cDNA of the SNCA gene in brain tissues of PD, DLB, and control samples using the PacBio Sequel system. The targeted full-length cDNA (Iso-Seq) data confirmed complex usage of known alternative start sites and variable 3textquoteright UTR lengths, as well as novel 5textquoteright starts and 3textquoteright ends not previously described. The targeted gDNA data allowed phasing of up to 81% of the ~114kb SNCA region, with the longest phased block excedding 54 kb. We demonstrate that long gDNA and cDNA reads have the potential to reveal long-range information not previously accessible using traditional sequencing methods. This approach has a potential impact in studying disease risk genes such as SNCA, providing new insights into the genetic etiologies, including perturbations to the landscape the gene transcripts, of human complex diseases such as synucleinopathies.

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April 21, 2020  |  

Schizophrenia risk variants influence multiple classes of transcripts of sorting nexin 19 (SNX19).

Genome-wide association studies (GWAS) have identified many genomic loci associated with risk for schizophrenia, but unambiguous identification of the relationship between disease-associated variants and specific genes, and in particular their effect on risk conferring transcripts, has proven difficult. To better understand the specific molecular mechanism(s) at the schizophrenia locus in 11q25, we undertook cis expression quantitative trait loci (cis-eQTL) mapping for this 2 megabase genomic region using postmortem human brain samples. To comprehensively assess the effects of genetic risk upon local expression, we evaluated multiple transcript features: genes, exons, and exon-exon junctions in multiple brain regions-dorsolateral prefrontal cortex (DLPFC), hippocampus, and caudate. Genetic risk variants strongly associated with expression of SNX19 transcript features that tag multiple rare classes of SNX19 transcripts, whereas they only weakly affected expression of an exon-exon junction that tags the majority of abundant transcripts. The most prominent class of SNX19 risk-associated transcripts is predicted to be overexpressed, defined by an exon-exon splice junction between exons 8 and 10 (junc8.10) and that is predicted to encode proteins that lack the characteristic nexin C terminal domain. Risk alleles were also associated with either increased or decreased expression of multiple additional classes of transcripts. With RACE, molecular cloning, and long read sequencing, we found a number of novel SNX19 transcripts that further define the set of potential etiological transcripts. We explored epigenetic regulation of SNX19 expression and found that DNA methylation at CpG sites near the primary transcription start site and within exon 2 partially mediate the effects of risk variants on risk-associated expression. ATAC sequencing revealed that some of the most strongly risk-associated SNPs are located within a region of open chromatin, suggesting a nearby regulatory element is involved. These findings indicate a potentially complex molecular etiology, in which risk alleles for schizophrenia generate epigenetic alterations and dysregulation of multiple classes of SNX19 transcripts.

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April 21, 2020  |  

Gammaherpesvirus Readthrough Transcription Generates a Long Non-Coding RNA That Is Regulated by Antisense miRNAs and Correlates with Enhanced Lytic Replication In Vivo.

Gammaherpesviruses, including the human pathogens Epstein?Barr virus (EBV) and Kaposi’s sarcoma-associated herpesvirus (KSHV) are oncogenic viruses that establish lifelong infections in hosts and are associated with the development of lymphoproliferative diseases and lymphomas. Recent studies have shown that the majority of the mammalian genome is transcribed and gives rise to numerous long non-coding RNAs (lncRNAs). Likewise, the large double-stranded DNA virus genomes of herpesviruses undergo pervasive transcription, including the expression of many as yet uncharacterized lncRNAs. Murine gammaperherpesvirus 68 (MHV68, MuHV-4, ?HV68) is a natural pathogen of rodents, and is genetically and pathogenically related to EBV and KSHV, providing a highly tractable model for studies of gammaherpesvirus biology and pathogenesis. Through the integrated use of parallel data sets from multiple sequencing platforms, we previously resolved transcripts throughout the MHV68 genome, including at least 144 novel transcript isoforms. Here, we sought to molecularly validate novel transcripts identified within the M3/M2 locus, which harbors genes that code for the chemokine binding protein M3, the latency B cell signaling protein M2, and 10 microRNAs (miRNAs). Using strand-specific northern blots, we validated the presence of M3-04, a 3.91 kb polyadenylated transcript that initiates at the M3 transcription start site and reads through the M3 open reading frame (ORF), the M3 poly(a) signal sequence, and the M2 ORF. This unexpected transcript was solely localized to the nucleus, strongly suggesting that it is not translated and instead may function as a lncRNA. Use of an MHV68 mutant lacking two M3-04-antisense pre-miRNA stem loops resulted in highly increased expression of M3-04 and increased virus replication in the lungs of infected mice, demonstrating a key role for these RNAs in regulation of lytic infection. Together these findings suggest the possibility of a tripartite regulatory relationship between the lncRNA M3-04, antisense miRNAs, and the latency gene M2.

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April 21, 2020  |  

Single-Molecule Sequencing: Towards Clinical Applications.

In the past several years, single-molecule sequencing platforms, such as those by Pacific Biosciences and Oxford Nanopore Technologies, have become available to researchers and are currently being tested for clinical applications. They offer exceptionally long reads that permit direct sequencing through regions of the genome inaccessible or difficult to analyze by short-read platforms. This includes disease-causing long repetitive elements, extreme GC content regions, and complex gene loci. Similarly, these platforms enable structural variation characterization at previously unparalleled resolution and direct detection of epigenetic marks in native DNA. Here, we review how these technologies are opening up new clinical avenues that are being applied to pathogenic microorganisms and viruses, constitutional disorders, pharmacogenomics, cancer, and more.Copyright © 2018 Elsevier Ltd. All rights reserved.

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April 21, 2020  |  

Improved annotation of the domestic pig genome through integration of Iso-Seq and RNA-seq data.

Our understanding of the pig transcriptome is limited. RNA transcript diversity among nine tissues was assessed using poly(A) selected single-molecule long-read isoform sequencing (Iso-seq) and Illumina RNA sequencing (RNA-seq) from a single White cross-bred pig. Across tissues, a total of 67,746 unique transcripts were observed, including 60.5% predicted protein-coding, 36.2% long non-coding RNA and 3.3% nonsense-mediated decay transcripts. On average, 90% of the splice junctions were supported by RNA-seq within tissue. A large proportion (80%) represented novel transcripts, mostly produced by known protein-coding genes (70%), while 17% corresponded to novel genes. On average, four transcripts per known gene (tpg) were identified; an increase over current EBI (1.9 tpg) and NCBI (2.9 tpg) annotations and closer to the number reported in human genome (4.2 tpg). Our new pig genome annotation extended more than 6000 known gene borders (5′ end extension, 3′ end extension, or both) compared to EBI or NCBI annotations. We validated a large proportion of these extensions by independent pig poly(A) selected 3′-RNA-seq data, or human FANTOM5 Cap Analysis of Gene Expression data. Further, we detected 10,465 novel genes (81% non-coding) not reported in current pig genome annotations. More than 80% of these novel genes had transcripts detected in >?1 tissue. In addition, more than 80% of novel intergenic genes with at least one transcript detected in liver tissue had H3K4me3 or H3K36me3 peaks mapping to their promoter and gene body, respectively, in independent liver chromatin immunoprecipitation data. These validated results show significant improvement over current pig genome annotations.

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