Nanopore sequencing reveals TACC2 locus complexity and diversity of isoforms transcribed from an intronic promoter.
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ABSTRACT:
SUBMITTER: Ito Y
PROVIDER: S-EPMC8087818 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundNonsense-mediated mRNA decay (NMD) is a eukaryotic, translation-dependent degradation pathway that targets mRNAs with premature termination codons and also regulates the expression of some mRNAs that encode full-length proteins. Although many genes express NMD-sensitive transcripts, identifying them based on short-read sequencing data remains a challenge.ResultsTo identify and analyze endogenous targets of NMD, we apply cDNA Nanopore sequencing and short-read sequencing to human cells with varying expression levels of NMD factors. Our approach detects full-length NMD substrates that are highly unstable and increase in levels or even only appear when NMD is inhibited. Among the many new NMD-targeted isoforms that our analysis identifies, most derive from alternative exon usage. The isoform-aware analysis reveals many genes with significant changes in splicing but no significant changes in overall expression levels upon NMD knockdown. NMD-sensitive mRNAs have more exons in the 3΄UTR and, for those mRNAs with a termination codon in the last exon, the length of the 3΄UTR per se does not correlate with NMD sensitivity. Analysis of splicing signals reveals isoforms where NMD has been co-opted in the regulation of gene expression, though the main function of NMD seems to be ridding the transcriptome of isoforms resulting from spurious splicing events.ConclusionsLong-read sequencing enables the identification of many novel NMD-sensitive mRNAs and reveals both known and unexpected features concerning their biogenesis and their biological role. Our data provide a highly valuable resource of human NMD transcript targets for future genomic and transcriptomic applications.
| S-EPMC8361881 | biostudies-literature
Project description:We recently reported the association of the PCSK6 gene with handedness through a quantitative genome-wide association study (GWAS; P < 0.5 × 10(-8)) for a relative hand skill measure in individuals with dyslexia. PCSK6 activates Nodal, a morphogen involved in regulating left-right body axis determination. Therefore, the GWAS data suggest that the biology underlying the patterning of structural asymmetries may also contribute to behavioural laterality, e.g. handedness. The association is further supported by an independent study reporting a variable number tandem repeat (VNTR) within the same PCSK6 locus to be associated with degree of handedness in a general population cohort. Here, we have conducted a functional analysis of the PCSK6 locus combining further genetic analysis, in silico predictions and molecular assays. We have shown that the previous GWAS signal was not tagging a VNTR effect, suggesting that the two markers have independent effects. We demonstrated experimentally that one of the top GWAS-associated markers, rs11855145, directly alters the binding site for a nuclear factor. Furthermore, we have shown that the predicted regulatory region adjacent to rs11855415 acts as a bidirectional promoter controlling the expression of novel RNA transcripts. These include both an antisense long non-coding RNA (lncRNA) and a short PCSK6 isoform predicted to be coding. This is the first molecular characterization of a handedness-associated locus that supports the role of common variants in non-coding sequences in influencing complex phenotypes through gene expression regulation.
| S-EPMC4986331 | biostudies-literature
Project description:We developed a single-cell approach to detect CRISPR-modified mRNA transcript structures. This method assesses how genetic variants at splicing sites and splicing factors contribute to alternative mRNA isoforms. We determine how alternative splicing is regulated by editing target exon-intron segments or splicing factors by CRISPR-Cas9 and their consequences on transcriptome profile. Our method combines long-read sequencing to characterize the transcript structure and short-read sequencing to match the single-cell gene expression profiles and gRNA sequence and therefore provides targeted genomic edits and transcript isoform structure detection at single-cell resolution.
| S-EPMC8647366 | biostudies-literature
Project description:IntroductionWhile tens of thousands of HLA alleles have been identified by DNA sequencing, the contribution of alternative splicing to HLA diversity is not well characterized. In this study, we sought to determine if long-read sequencing could be used to accurately quantify allele-specific HLA transcripts in primary human lymphocytes.MethodscDNA libraries were prepared from peripheral blood lymphocytes from 12 donors and sequenced by nanopore long-read sequencing. HLA reads were aligned to donor-specific reference sequences based on the known type of each donor. Allele-specific exon utilization was calculated as the proportion of reads aligning to each allele containing known exons, and transcript isoforms were quantified based on patterns of exon utilization within individual reads.ResultsSplice variants were rare among class I HLA genes (median exon retention rate 99%-100%), except for several HLA-C alleles with exon 5 spliced out of up to 15% of reads. Splice variants were also rare among class II HLA genes (median exon retention rate 98%-100%), except for HLA-DQB1. Consistent with previous work, exon 5 of HLA-DQB1 was spliced out in alleles with a mutated splice acceptor site at rs28688207. Surprisingly, a 28% loss of exon 5 was also observed in HLA-DQB1 alleles with an intact splice acceptor site at rs28688207.DiscussionWe describe a simple bioinformatic workflow to quantify allele-specific expression of HLA transcript isoforms. Further studies are warranted to characterize the repertoire of HLA transcripts expressed in different cell types and tissues across diverse populations.
| S-EPMC10471969 | biostudies-literature
Project description:The use of alternative transcription start or termination sites (aTSS or aTTS) as well as alternative splicing (AS) produce diverse transcript isoforms, playing indispensable roles in the plant development and environmental adaptations. Despite the advances in the finding of the genome-wide alternatively spliced genes in strawberry, it remains unexplored how AS responds to the developmental cues and what relevance do these outcomes have to the gene function. In this study, we have systematically investigated the transcriptome complexity using long-read Oxford Nanopore Technologies along the four successive developmental stages. The full-length cDNA sequencing results unraveled thousands of previously unexplored transcript isoforms raised from aTSS, aTTS, and AS. The relative contributions of these three processes to the complexity of strawberry fruit transcripts were compared. The aTSS and aTTS were more abundant than the AS. Differentially expressed transcripts unraveled the key transitional role of the white fruit stage. Isoform switches of transcripts from 757 genes were observed. They were associated with protein-coding potential change and domain gain or loss as the main consequences. Those genes with switched isoforms take part in the key processes of maturation in the late stages. A case study using yeast two hybrid analysis supported the functional divergence of the two isoforms of the B-box protein 22. Our results provided a new comprehensive overview of the dynamic transcriptomic landscape during strawberry fruit development and maturation.
| S-EPMC9326444 | biostudies-literature
Project description:Nonsense-mediated mRNA decay (NMD) is a eukaryotic, translation-dependent degradation pathway that targets mRNAs with premature termination codons and also regulates the expression of some mRNAs that encode full-length proteins. Although many genes express NMD-sensitive transcripts, identifying them based on short-read sequencing data remains a challenge. To identify and analyze endogenous targets of NMD, we applied cDNA Nanopore sequencing and short-read sequencing to human cells with varying expression levels of NMD factors. Our approach detects full-length NMD substrates that are highly unstable and increase in levels or even only appear when NMD is inhibited. Among the many new NMD-targeted isoforms that our analysis identified, most derive from alternative exon usage. The isoform-aware analysis revealed many genes with significant changes in splicing but no significant changes in overall expression levels upon NMD knockdown. NMD-sensitive mRNAs have more exons in the 3΄UTR and for mRNAs with a termination codon in the last exon. The length of the 3΄UTR per se does not correlate with NMD sensitivity. Analysis of splicing signals reveals isoforms where NMD has been co-opted in the regulation of gene expression, but a main function is most likely to rid the transcriptome of isoforms resulting from spurious splicing events. Long-read sequencing enabled the identification of many novel NMD-sensitive mRNAs and revealed both known and unexpected features concerning their biogenesis and their biological role. Our data provide a highly valuable resource of human NMD transcript targets for future genomic and transcriptomic applications.
2021-07-20 | E-MTAB-10452 | biostudies-arrayexpress
Project description:BackgroundHigh sequence identity between segmental duplications (SDs) can facilitate copy number variants (CNVs) via non-allelic homologous recombination (NAHR). These CNVs are one of the fundamental causes of genomic disorders such as the 3q29 deletion syndrome (del3q29S). There are 21 protein-coding genes lost or gained as a result of such recurrent 1.6-Mbp deletions or duplications, respectively, in the 3q29 locus. While NAHR plays a role in CNV occurrence, the factors that increase the risk of NAHR at this particular locus are not well understood.MethodsWe employed an optical genome mapping technique to characterize the 3q29 locus in 161 unaffected individuals, 16 probands with del3q29S and their parents, and 2 probands with the 3q29 duplication syndrome (dup3q29S). Long-read sequencing-based haplotype resolved de novo assemblies from 44 unaffected individuals, and 1 trio was used for orthogonal validation of haplotypes and deletion breakpoints.ResultsIn total, we discovered 34 haplotypes, of which 19 were novel haplotypes. Among these 19 novel haplotypes, 18 were detected in unaffected individuals, while 1 novel haplotype was detected on the parent-of-origin chromosome of a proband with the del3q29S. Phased assemblies from 44 unaffected individuals enabled the orthogonal validation of 20 haplotypes. In 89% (16/18) of the probands, breakpoints were confined to paralogous copies of a 20-kbp segment within the 3q29 SDs. In one del3q29S proband, the breakpoint was confined to a 374-bp region using long-read sequencing. Furthermore, we categorized del3q29S cases into three classes and dup3q29S cases into two classes based on breakpoints. Finally, we found no evidence of inversions in parent-of-origin chromosomes.ConclusionsWe have generated the most comprehensive haplotype map for the 3q29 locus using unaffected individuals, probands with del3q29S or dup3q29S, and available parents, and also determined the deletion breakpoint to be within a 374-bp region in one proband with del3q29S. These results should provide a better understanding of the underlying genetic architecture that contributes to the etiology of del3q29S and dup3q29S.
| S-EPMC10170684 | biostudies-literature
Project description:Alternative splicing is a widespread phenomenon in metazoans by which single genes are able to produce multiple isoforms of the gene product. However, this has been poorly characterized in apicomplexans, a major phylum of some of the most important global parasites. Efforts have been hampered by atypical transcriptomic features, such as the high AU content of Plasmodium RNA, but also the limitations of short-read sequencing in deciphering complex splicing events. In this study, we utilized the long read direct RNA sequencing platform developed by Oxford Nanopore Technologies to survey the alternative splicing landscape of Toxoplasma gondii and Plasmodium falciparum We find that while native RNA sequencing has a reduced throughput, it allows us to obtain full-length or nearly full-length transcripts with comparable quantification to Illumina sequencing. By comparing these data with available gene models, we find widespread alternative splicing, particularly intron retention, in these parasites. Most of these transcripts contain premature stop codons, suggesting that in these parasites, alternative splicing represents a pathway to transcriptomic diversity, rather than expanding proteomic diversity. Moreover, alternative splicing rates are comparable between parasites, suggesting a shared splicing machinery, despite notable transcriptomic differences between the parasites. This study highlights a strategy in using long-read sequencing to understand splicing events at the whole-transcript level and has implications in the future interpretation of transcriptome sequencing studies.IMPORTANCE We have used a novel nanopore sequencing technology to directly analyze parasite transcriptomes. The very long reads of this technology reveal the full-length genes of the parasites that cause malaria and toxoplasmosis. Gene transcripts must be processed in a process called splicing before they can be translated to protein. Our analysis reveals that these parasites very frequently only partially process their gene products, in a manner that departs dramatically from their human hosts.
| S-EPMC8561664 | biostudies-literature
Project description:The miniaturized and portable DNA sequencer MinION™ has demonstrated great potential in different analyses such as genome-wide sequencing, pathogen outbreak detection and surveillance, human genome variability, and microbial diversity. In this study, we tested the ability of the MinION™ platform to perform long amplicon sequencing in order to design new approaches to study microbial diversity using a multi-locus approach. After compiling a robust database by parsing and extracting the rrn bacterial region from more than 67000 complete or draft bacterial genomes, we demonstrated that the data obtained during sequencing of the long amplicon in the MinION™ device using R9 and R9.4 chemistries were sufficient to study 2 mock microbial communities in a multiplex manner and to almost completely reconstruct the microbial diversity contained in the HM782D and D6305 mock communities. Although nanopore-based sequencing produces reads with lower per-base accuracy compared with other platforms, we presented a novel approach consisting of multi-locus and long amplicon sequencing using the MinION™ MkIb DNA sequencer and R9 and R9.4 chemistries that help to overcome the main disadvantage of this portable sequencing platform. Furthermore, the nanopore sequencing library, constructed with the last releases of pore chemistry (R9.4) and sequencing kit (SQK-LSK108), permitted the retrieval of the higher level of 1D read accuracy sufficient to characterize the microbial species present in each mock community analysed. Improvements in nanopore chemistry, such as minimizing base-calling errors and new library protocols able to produce rapid 1D libraries, will provide more reliable information in the near future. Such data will be useful for more comprehensive and faster specific detection of microbial species and strains in complex ecosystems.
| S-EPMC5534310 | biostudies-literature
Project description:Facioscapulohumeral muscular dystrophy (FSHD) is an inherited muscle disease caused by misexpression of the DUX4 gene in skeletal muscle. DUX4 is a transcription factor, which is normally expressed in the cleavage-stage embryo and regulates gene expression involved in early embryonic development. Recent studies revealed that DUX4 also activates the transcription of repetitive elements such as endogenous retroviruses (ERVs), mammalian apparent long terminal repeat (LTR)-retrotransposons and pericentromeric satellite repeats (Human Satellite II). DUX4-bound ERV sequences also create alternative promoters for genes or long non-coding RNAs, producing fusion transcripts. To further understand transcriptional regulation by DUX4, we performed nanopore long-read direct RNA sequencing (dRNA-seq) of human muscle cells induced by DUX4, because long reads show whole isoforms with greater confidence. We successfully detected differential expression of known DUX4-induced genes and discovered 61 differentially expressed repeat loci, which are near DUX4-ChIP peaks. We also identified 247 gene-ERV fusion transcripts, of which 216 were not reported previously. In addition, long-read dRNA-seq clearly shows that RNA splicing is a common event in DUX4-activated ERV transcripts. Long-read analysis showed non-LTR transposons including Alu elements are also transcribed from LTRs. Our findings revealed further complexity of DUX4-induced ERV transcripts. This catalogue of DUX4-activated repetitive elements may provide useful information to elucidate the pathology of FSHD. Also, our results indicate that nanopore dRNA-seq has complementary strengths to conventional short-read complementary DNA sequencing.
| S-EPMC8120133 | biostudies-literature