Genome research最新文献

Despite recent advances made toward improving the efficacy of lentiviral gene therapies, a sizeable proportion of produced vector contains an incomplete and thus potentially nonfunctional RNA genome. This can undermine gene delivery by the lentivirus as well as increase manufacturing costs and must be improved to facilitate the widespread clinical implementation of lentiviral gene therapies. Here, we compare three long-read sequencing technologies for their ability to detect issues in vector design and determine nanopore direct RNA sequencing to be the most powerful. We show how this approach identifies and quantifies incomplete RNA caused by cryptic splicing and polyadenylation sites, including a potential cryptic polyadenylation site in the widely used Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE). Using artificial polyadenylation of the lentiviral RNA, we also identify multiple hairpin-associated truncations in the analyzed lentiviral vectors (LVs), which account for most of the detected RNA fragments. Finally, we show that these insights can be used for the optimization of LV design. In summary, nanopore direct RNA sequencing is a powerful tool for the quality control and optimization of LVs, which may help to improve lentivirus manufacturing and thus the development of higher quality lentiviral gene therapies.

DNA modifications in bacteria present diverse types and distributions, playing crucial functional roles. Current methods for detecting bacterial DNA modifications via nanopore sequencing typically involve comparing raw current signals to a methylation-free control. In this study, we found that bacterial DNA modification induces errors in nanopore reads. And these errors are found only in one strand but not the other, showing a strand-specific bias. Leveraging this discovery, we developed Hammerhead, a pioneering pipeline designed for de novo methylation discovery that circumvents the necessity of raw signal inference and a methylation-free control. The majority (14 out of 16) of the identified motifs can be validated by raw signal comparison methods or by identifying corresponding methyltransferases in bacteria. Additionally, we included a novel polishing strategy employing duplex reads to correct modification-induced errors in bacterial genome assemblies, achieving a reduction of over 85% in such errors. In summary, Hammerhead enables users to effectively locate bacterial DNA methylation sites from nanopore FASTQ/FASTA reads, thus holds promise as a routine pipeline for a wide range of nanopore sequencing applications, such as genome assembly, metagenomic binning, decontaminating eukaryotic genome assemblies, and functional analysis for DNA modifications.

Long-read sequencing technologies have improved the contiguity and, as a result, the quality of genome assemblies by generating reads long enough to span and resolve complex or repetitive regions of the genome. Several groups have shown the power of long reads in detecting thousands of genomic and epigenomic features that were previously missed by short-read sequencing approaches. While these studies demonstrate how long reads can help resolve repetitive and complex regions of the genome, they also highlight the throughput and coverage requirements needed to accurately resolve variant alleles across large populations using these platforms. At the time of this review, whole-genome long-read sequencing is more expensive than short-read sequencing on the highest throughput short-read instruments; thus, achieving sufficient coverage to detect low-frequency variants (such as somatic variation) in heterogenous samples remains challenging. Targeted sequencing, on the other hand, provides the depth necessary to detect these low-frequency variants in heterogeneous populations. Here, we review currently used and recently developed targeted sequencing strategies that leverage existing long-read technologies to increase the resolution with which we can look at nucleic acids in a variety of biological contexts.

Fewer than half of individuals with a suspected Mendelian or monogenic condition receive a precise molecular diagnosis after comprehensive clinical genetic testing. Improvements in data quality and costs have heightened interest in using long-read sequencing (LRS) to streamline clinical genomic testing, but the absence of control data sets for variant filtering and prioritization has made tertiary analysis of LRS data challenging. To address this, the 1000 Genomes Project (1KGP) Oxford Nanopore Technologies Sequencing Consortium aims to generate LRS data from at least 800 of the 1KGP samples. Our goal is to use LRS to identify a broader spectrum of variation so we may improve our understanding of normal patterns of human variation. Here, we present data from analysis of the first 100 samples, representing all 5 superpopulations and 19 subpopulations. These samples, sequenced to an average depth of coverage of 37× and sequence read N50 of 54 kbp, have high concordance with previous studies for identifying single nucleotide and indel variants outside of homopolymer regions. Using multiple structural variant (SV) callers, we identify an average of 24,543 high-confidence SVs per genome, including shared and private SVs likely to disrupt gene function as well as pathogenic expansions within disease-associated repeats that were not detected using short reads. Evaluation of methylation signatures revealed expected patterns at known imprinted loci, samples with skewed X-inactivation patterns, and novel differentially methylated regions. All raw sequencing data, processed data, and summary statistics are publicly available, providing a valuable resource for the clinical genetics community to discover pathogenic SVs.

Transmission of carbapenem-resistant Enterobacterales (CRE) in hospitals has been shown to occur through complex, multifarious networks driven by both clonal spread and horizontal transfer mediated by plasmids and other mobile genetic elements. We performed nanopore long-read sequencing on CRE isolates from a large urban hospital system to determine the overall contribution of plasmids to CRE transmission and identify specific plasmids implicated in the spread of bla _KPC (the Klebsiella pneumoniae carbapenemase [KPC] gene). Six hundred and five CRE isolates collected between 2009 and 2018 first underwent Illumina sequencing for genome-wide genotyping; 435 bla _KPC-positive isolates were then successfully nanopore sequenced to generate hybrid assemblies including circularized bla _KPC-harboring plasmids. Phylogenetic analysis and Mash clustering were used to define putative clonal and plasmid transmission clusters, respectively. Overall, CRE isolates belonged to 96 multilocus sequence types (STs) encoding bla _KPC on 447 plasmids which formed 54 plasmid clusters. We found evidence for clonal transmission in 66% of CRE isolates, over half of which belonged to four clades comprising K. pneumoniae ST258. Plasmid-mediated acquisition of bla _KPC occurred in 23%-27% of isolates. While most plasmid clusters were small, several plasmids were identified in multiple different species and STs, including a highly promiscuous IncN plasmid and an IncF plasmid putatively spreading bla _KPC from ST258 to other clones. Overall, this points to both the continued dominance of K. pneumoniae ST258 and the dissemination of bla _KPC across clones and species by diverse plasmid backbones. These findings support integrating long-read sequencing into genomic surveillance approaches to detect the hitherto silent spread of carbapenem resistance driven by mobile plasmids.

The combination of ultra-long (UL) Oxford Nanopore Technologies (ONT) sequencing reads with long, accurate Pacific Bioscience (PacBio) High Fidelity (HiFi) reads has enabled the completion of a human genome and spurred similar efforts to complete the genomes of many other species. However, this approach for complete, "telomere-to-telomere" genome assembly relies on multiple sequencing platforms, limiting its accessibility. ONT "Duplex" sequencing reads, where both strands of the DNA are read to improve quality, promise high per-base accuracy. To evaluate this new data type, we generated ONT Duplex data for three widely studied genomes: human HG002, Solanum lycopersicum Heinz 1706 (tomato), and Zea mays B73 (maize). For the diploid, heterozygous HG002 genome, we also used "Pore-C" chromatin contact mapping to completely phase the haplotypes. We found the accuracy of Duplex data to be similar to HiFi sequencing, but with read lengths tens of kilobases longer, and the Pore-C data to be compatible with existing diploid assembly algorithms. This combination of read length and accuracy enables the construction of a high-quality initial assembly, which can then be further resolved using the UL reads, and finally phased into chromosome-scale haplotypes with Pore-C. The resulting assemblies have a base accuracy exceeding 99.999% (Q50) and near-perfect continuity, with most chromosomes assembled as single contigs. We conclude that ONT sequencing is a viable alternative to HiFi sequencing for de novo genome assembly, and provides a multirun single-instrument solution for the reconstruction of complete genomes.

Alternative splicing (AS) alters the cis-regulatory landscape of mRNA isoforms, leading to transcripts with distinct localization, stability, and translational efficiency. To rigorously investigate mRNA isoform-specific ribosome association, we generated subcellular fractionation and sequencing (Frac-seq) libraries using both conventional short reads and long reads from human embryonic stem cells (ESCs) and neural progenitor cells (NPCs) derived from the same ESCs. We performed de novo transcriptome assembly from high-confidence long reads from cytosolic, monosomal, light, and heavy polyribosomal fractions and quantified their abundance using short reads from their respective subcellular fractions. Thousands of transcripts in each cell type exhibited association with particular subcellular fractions relative to the cytosol. Of the multi-isoform genes, 27% and 19% exhibited significant differential isoform sedimentation in ESCs and NPCs, respectively. Alternative promoter usage and internal exon skipping accounted for the majority of differences between isoforms from the same gene. Random forest classifiers implicated coding sequence (CDS) and untranslated region (UTR) lengths as important determinants of isoform-specific sedimentation profiles, and motif analyses reveal potential cell type-specific and subcellular fraction-associated RNA-binding protein signatures. Taken together, our data demonstrate that alternative mRNA processing within the CDS and UTRs impacts the translational control of mRNA isoforms during stem cell differentiation, and highlight the utility of using a novel long-read sequencing-based method to study translational control.

Genomic resources are important for evaluating genetic diversity and supporting conservation efforts. The garden dormouse (Eliomys quercinus) is a small rodent that has experienced one of the most severe modern population declines in Europe. We present a high-quality haplotype-resolved reference genome for the garden dormouse, and combine comprehensive short and long-read transcriptomics data sets with homology-based methods to generate a highly complete gene annotation. Demographic history analysis of the genome reveal a sharp population decline since the last interglacial, indicating an association between colder climates and population declines before anthropogenic influence. Using our genome and genetic data from 100 individuals, largely sampled in a citizen-science project across the contemporary range, we conduct the first population genomic analysis for this species. We find clear evidence for population structure across the species' core Central European range. Notably, our data show that the Alpine population, characterized by strong differentiation and reduced genetic diversity, is reproductively isolated from other regions and likely represents a differentiated evolutionary significant unit (ESU). The predominantly declining Eastern European populations also show signs of recent isolation, a pattern consistent with a range expansion from Western to Eastern Europe during the Holocene, leaving relict populations now facing local extinction. Overall, our findings suggest that garden dormouse conservation may be enhanced in Europe through the designation of ESUs.