Biology subjects

Timp, W.

Publications and source records attributed to Timp, W..

48 records · Page 3Linked to original sources

bioRxiv2019

Comprehensive analysis of structural variants in breast cancer genomes using single molecule sequencing

Improved identification of structural variants (SVs) in cancer can lead to more targeted and effective treatment options as well as advance our basic understanding of disease progression. We performed whole genome sequencing of the SKBR3 breast cancer cell-line and patient-derived tumor and normal organoids from two breast cancer patients using 10X/Illumina, PacBio, and Oxford Nanopore sequencing. We then inferred SVs and large-scale allele-specific copy number variants (CNVs) using an ensemble of methods. Our findings demonstrate that long-read sequencing allows for substantially more accurate and sensitive SV detection, with between 90% and 95% of variants supported by each long-read technology also supported by the other. We also report high accuracy for long-reads even at relatively low coverage (25x-30x). Furthermore, we inferred karyotypes from these data using our enhanced RCK algorithm to present a more accurate representation of the mutated cancer genomes, and find hundreds of variants affecting known cancer-related genes detectable only through long-read sequencing. These findings highlight the need for long-read sequencing of cancer genomes for the precise analysis of their genetic instability.

bioinformatics↗

bioRxiv2019

High-quality chromosome-scale assembly of the walnut (Juglans regia L) reference genome

The release of the first reference genome of walnut (Juglans regia L.) enabled many achievements in the characterization of walnut genetic and functional variation. However, it is highly fragmented, preventing the integration of genetic, transcriptomic, and proteomic information to fully elucidate walnut biological processes. Here we report the new chromosome-scale assembly of the walnut reference genome (Chandler v2.0) obtained by combining Oxford Nanopore long-read sequencing with chromosome conformation capture (Hi-C) technology. Relative to the previous reference genome, the new assembly features an 84.4-fold increase in N50 size, and the full sequence of all 16 chromosomal pseudomolecules, nine of which present telomere sequences at both ends. Using full-length transcripts from single-molecule real-time sequencing, we predicted 40,491 gene models, with a mean gene length higher than the previous gene annotations. Most of the new protein-coding genes (90%) are full-length, which represents a significant improvement compared to Chandler v1.0 (only 48%). We then tested the potential impact of the new chromosome-level genome on different areas of walnut research. By studying the proteome changes occurring during catkin development, we observed that the virtual proteome obtained from Chandler v2.0 presents fewer artifacts than the previous reference genome, enabling the identification of a new potential pollen allergen in walnut. Also, the new chromosome-scale genome facilitates in-depth studies of intraspecies genetic diversity by revealing previously undetected autozygous regions in Chandler, likely resulting from inbreeding, and 195 genomic regions highly differentiated between Western and Eastern walnut cultivars. Overall, Chandler v2.0 is a valuable resource to understand and explore walnut biology better.

genomics↗

bioRxiv2019

Telomere-to-telomere assembly of a complete human X chromosome

After nearly two decades of improvements, the current human reference genome (GRCh38) is the most accurate and complete vertebrate genome ever produced. However, no one chromosome has been finished end to end, and hundreds of unresolved gaps persist 1,2. The remaining gaps include ribosomal rDNA arrays, large near-identical segmental duplications, and satellite DNA arrays. These regions harbor largely unexplored variation of unknown consequence, and their absence from the current reference genome can lead to experimental artifacts and hide true variants when re-sequencing additional human genomes. Here we present a de novo human genome assembly that surpasses the continuity of GRCh38 2, along with the first gapless, telomere-to-telomere assembly of a human chromosome. This was enabled by high-coverage, ultra-long-read nanopore sequencing of the complete hydatidiform mole CHM13 genome, combined with complementary technologies for quality improvement and validation. Focusing our efforts on the human X chromosome 3, we reconstructed the [~]2.8 megabase centromeric satellite DNA array and closed all 29 remaining gaps in the current reference, including new sequence from the human pseudoautosomal regions and cancer-testis ampliconic gene families (CT-X and GAGE). This complete chromosome X, combined with the ultra-long nanopore data, also allowed us to map methylation patterns across complex tandem repeats and satellite arrays for the first time. These results demonstrate that finishing the human genome is now within reach and will enable ongoing efforts to complete the remaining human chromosomes.

bioinformatics↗

bioRxiv2019

Targeting destabilized DNA G-quadruplexes and aberrant splicing in drug-resistant glioblastoma

Temozolomide (TMZ) is a chemotherapeutic agent that has been the first-line standard of care for the aggressive brain cancer glioblastoma (GBM) since 2005. Though initially beneficial, TMZ- resistance is universal and second-line interventions are an unmet clinical need. Here we took advantage the mechanism of action of TMZ to target guanines (G) and investigated G-rich g- quadruplex (G4) and splice site changes that occur upon TMZ-resistance. We report TMZ-resistant GBM has guanine mutations that disrupt the G-rich DNA G4s and splice sites that lead to deregulated alternative splicing. These alterations create vulnerabilities, which are selectively targeted by either the G4 stabilizing drug TMPyP4 or a novel splicing kinase inhibitor of cdc2- like kinase. Finally, we show that the G4 and RNA-binding protein EWSR1 aggregates in the cytoplasm in TMZ-resistant GBM cells and patient samples. Together, our findings provide insight into targetable vulnerabilities of TMZ-resistant GBM and present cytoplasmic EWSR1 as a putative biomarker. TeaserTargeting temozolomide mutations in drug resistant glioma via g-quadruplex and splicing modulators with a putative biomarker.

cancer biology↗

bioRxiv2019

Targeted Nanopore Sequencing with Cas9 for studies of methylation, structural variants and mutations

Nanopore sequencing technology can rapidly and directly interrogate native DNA molecules. Often we are interested only in interrogating specific areas at high depth, but conventional enrichment methods have thus far proved unsuitable for long reads1. Existing strategies are currently limited by high input DNA requirements, low yield, short (<5kb) reads, time-intensive protocols, and/or amplification or cloning (losing base modification information). In this paper, we describe a technique utilizing the ability of Cas9 to introduce cuts at specific locations and ligating nanopore sequencing adaptors directly to those sites, a method we term nanopore Cas9 Targeted-Sequencing (nCATS).\n\nWe have demonstrated this using an Oxford Nanopore MinION flow cell (Capacity >10Gb+) to generate a median 165X coverage at 10 genomic loci with a median length of 18kb, representing a several hundred-fold improvement over the 2-3X coverage achieved without enrichment. We performed a pilot run on the smaller Flongle flow cell (Capacity ~1Gb), generating a median coverage of 30X at 11 genomic loci with a median length of 18kb. Using panels of guide RNAs, we show that the high coverage data from this method enables us to (1) profile DNA methylation patterns at cancer driver genes, (2) detect structural variations at known hot spots, and (3) survey for the presence of single nucleotide mutations. Together, this provides a low-cost method that can be applied even in low resource settings to directly examine cellular DNA. This technique has extensive clinical applications for assessing medically relevant genes and has the versatility to be a rapid and comprehensive diagnostic tool. We demonstrate applications of this technique by examining the well-characterized GM12878 cell line as well as three breast cell lines (MCF-10A, MCF-7, MDA-MB-231) with varying tumorigenic potential as a model for cancer.\n\nContributionsTG and WT constructed the study. TG performed the experiments. TG, IL, and FS analyzed the data. TG, JG, ER, RB and AH and developed the method. TG and WT wrote the paper

genomics↗

bioRxiv2019

The full-length transcriptome of C. elegans using direct RNA sequencing

Current transcriptome annotations have largely relied on short read lengths intrinsic to most widely used high-throughput cDNA sequencing technologies. For example, in the annotation of the Caenorhabditis elegans transcriptome, more than half of the transcript isoforms lack full-length support and instead rely on inference from short reads that do not span the full length of the isoform. We applied nanopore-based direct RNA sequencing to characterize the developmental polyadenylated transcriptome of C. elegans. Taking advantage of long reads spanning the full length of mRNA transcripts, we provide support for 20,902 splice isoforms across 14,115 genes, without the need for computational reconstruction of gene models. Of the isoforms identified, 2,188 are novel splice isoforms not present in the Wormbase WS265 annotation. Furthermore, we identified 16,325 3 untranslated region (3UTR) isoforms, 2,304 of which are novel and do not fall within 10 bp of existing 3UTR datasets and annotations. Combining 3UTRs and splice isoforms we identified 25,944 full-length isoforms. We also determined that poly(A) tail lengths of transcripts vary across development, as do the strengths of previously reported correlations between poly(A) tail length and expression level, and poly(A) tail length and 3UTR length. Finally, we have formatted this data as a publically accessible track hub, enabling researchers to explore this dataset easily in a genome browser.

genomics↗

bioRxiv2018

Simultaneous profiling of chromatin accessibility and methylation on human cell lines with nanopore sequencing

Understanding how the genome and the epigenome work together to control gene transcription has applications in our understanding of diseases such as human cancer. In this study, we combine the ability of NOMe-seq to simultaneously evaluate CpG methylation and chromatin accessibility, with long-read nanopore sequencing technology, a method we call nanoNOMe. We generated >60Gb whole-genome nanopore sequencing data for each of four human cell lines (GM12878, MCF-10A, MCF-7, MDA-MB-231) including repetitive regions inaccessible by short read sequencing. Using the long reads, we find that we can observe phased methylation and chromatin accessibility, large scale pattern changes, and genetic changes such as structural variations from a single assay.

genomics↗

bioRxiv2018

Nanopore native RNA sequencing of a human poly(A) transcriptome

High throughput cDNA sequencing technologies have dramatically advanced our understanding of transcriptome complexity and regulation. However, these methods lose information contained in biological RNA because the copied reads are often short and because modifications are not carried forward in cDNA. We address these limitations using a native poly(A) RNA sequencing strategy developed by Oxford Nanopore Technologies (ONT). Our study focused on poly(A) RNA from the human cell line GM12878, generating 9.9 million aligned sequence reads. These native RNA reads had an aligned N50 length of 1294 bases, and a maximum aligned length of over 21,000 bases. A total of 78,199 high-confidence isoforms were identified by combining long nanopore reads with short higher accuracy Illumina reads. We describe strategies for assessing 3' poly(A) tail length, base modifications and transcript haplotypes from nanopore RNA data. Together, these nanopore-based techniques are poised to deliver new insights into RNA biology.\n\nDISCLOSURESMA holds shares in Oxford Nanopore Technologies (ONT). MA is a paid consultant to ONT. REW, WT, TG, JRT, JQ, NJL, JTS, NS, AB, MA, HEO, MJ, and ML received reimbursement for travel, accommodation and conference fees to speak at events organised by ONT. NL has received an honorarium to speak at an ONT company meeting. WT has two patents (8,748,091 and 8,394,584) licensed to Oxford Nanopore. JTS, ML and MA received research funding from ONT.

genomics↗

bioRxiv2018

Assessing 16S marker gene survey data analysis methods using mixtures of human stool sample DNA extracts.

BackgroundAnalysis of 16S rRNA marker-gene surveys, used to characterize prokaryotic microbial communities, may be performed by numerous bioinformatic pipelines and downstream analysis methods. However, there is limited guidance on how to decide between methods, appropriate data sets and statistics for assessing these methods are needed. We developed a mixture dataset with real data complexity and an expected value for assessing 16S rRNA bioinformatic pipelines and downstream analysis methods. We generate an assessment dataset using a two-sample titration mixture design. The sequencing data were processed using multiple bioinformatic pipelines, i) DADA2 a sequence inference method, ii) Mothur a de novo clustering method, and iii) QIIME with open-reference clustering. The mixture dataset was used to qualitatively and quantitatively assess count tables generated using the pipelines.\n\nResultsThe qualitative assessment was used to evalute features only present in unmixed samples and titrations. The abundance of Mothur and QIIME features specific to unmixed samples and titrations were explained by sampling alone. However, for DADA2 over a third of the unmixed sample and titration specific feature abundance could not be explained by sampling alone. The quantitative assessment evaluated pipeline performance by comparing observed to expected relative and differential abundance values. Overall the observed relative abundance and differential abundance values were consistent with the expected values. Though outlier features were observed across all pipelines.\n\nConclusionsUsing a novel mixture dataset and assessment methods we quantitatively and qualitatively evaluated count tables generated using three bioinformatic pipelines. The dataset and methods developed for this study will serve as a valuable community resource for assessing 16S rRNA marker-gene survey bioinformatic methods.

bioinformatics↗

bioRxiv2018

Epigenetic changes induced by Bacteroides fragilis toxin

Enterotoxigenic Bacteroides fragilis (ETBF) is a gram negative, obligate anaerobe member of the gut microbial community in up to 40% of healthy individuals. This bacterium is found more frequently in people with colorectal cancer (CRC) and causes tumor formation in the distal colon of mice heterozygous for the adenomatous polyposis coli gene (Apc+/-); tumor formation is dependent on ETBF-secreted Bacteroides fragilis toxin (BFT). Though some of the immediate downstream effects of BFT on colon epithelial cells (CECs) are known, we still do not understand how this potent exotoxin causes changes in CECs that lead to tumor formation and growth. Because of the extensive data connecting alterations in the epigenome with tumor formation, initial experiments attempting to connect BFT-induced tumor formation with methylation in CECs have been performed, but the effect of BFT on other epigenetic processes, such as chromatin structure, remains unexplored. Here, the changes in chromatin accessibility (ATAC-seq) and gene expression (RNA-seq) induced by treatment of HT29/C1 cells with BFT for 24 and 48 hours is examined. Our data show that several genes are differentially expressed after BFT treatment and these changes correlate with changes in chromatin accessibility. Also, sites of increased chromatin accessibility are associated with a lower frequency of common single nucleotide variants (SNVs) in CRC and with a higher frequency of common differentially methylated regions (DMRs) in CRC. These data provide insight into the mechanisms by which BFT induces tumor formation. Further understanding of how BFT impacts nuclear structure and function in vivo is needed.\n\nImportanceColorectal cancer (CRC) is a major public health concern; there were approximately 135,430 new cases in 2017, and CRC is the second leading cause of cancer-related deaths for both men and women in the US (1). Many factors have been linked to CRC development, the most recent of which is the gut microbiome. Pre-clinical models support that enterotoxigenic Bacteroides fragilis (ETBF), among other bacteria, induce colon carcinogenesis. However, it remains unclear if the virulence determinants of any pro-carcinogenic colon bacterium induce DNA mutations or changes that initiate clonal CEC expansion. Using a reductionist model, we demonstrate that BFT rapidly alters chromatin structure and function consistent with capacity to contribute to CRC pathogenesis.

genomics↗

bioRxiv2017

First Draft Genome Sequence of the Pathogenic Fungus Lomentospora prolificans (formerly Scedosporium prolificans)

Here we describe the sequencing and assembly of the pathogenic fungus Lomentospora prolificans using a combination of short, highly accurate Illumina reads and additional coverage in very long Oxford Nanopore reads. The resulting assembly is highly contiguous, containing a total of 37,630,066 bp with over 98% of the sequence in just 26 scaffolds. Annotation identified 8,656 protein-coding genes. Pulsed-field gel analysis suggests that this organism contains at least 7 and possibly 11 chromosomes, the two longest of which have sizes corresponding closely to the sizes of the longest scaffolds, at 6.6 and 5.7 Mb.

microbiology↗

bioRxiv2017

Single molecule, full-length transcript sequencing provides insight into the extreme metabolism of ruby-throated hummingbird Archilochus colubris

Hummingbirds can support their high metabolic rates exclusively by oxidizing ingested sugars, which is unsurprising given their sugar-rich nectar diet and use of energetically expensive hovering flight. However, they cannot rely on dietary sugars as a fuel during fasting periods, such as during the night, at first light, or when undertaking long-distance migratory flights, and must instead rely exclusively on onboard lipids. This metabolic flexibility is remarkable both in that the birds can switch between exclusive use of each fuel type within minutes and in that de novo lipogenesis from dietary sugar precursors is the principle way in which fat stores are built, sometimes at exceptionally high rates, such as during the few days prior to a migratory flight. The hummingbird hepatopancreas is the principle location of de novo lipogenesis and likely plays a key role in fuel selection, fuel switching, and glucose homeostasis. Yet understanding how this tissue, and the whole organism, achieves and moderates high rates of energy turnover is hampered by a fundamental lack of information regarding how genes coding for relevant enzymes differ in their sequence, expression, and regulation in these unique animals. To address this knowledge gap, we generated a de novo transcriptome of the hummingbird liver using PacBio full-length cDNA sequencing (Iso-Seq), yielding a total of 8.6Gb of sequencing data, or 2.6M reads from 4 different size fractions. We analyzed data using the SMRTAnalysis v3.1 Iso-Seq pipeline, including classification of reads and clustering of isoforms (ICE) followed by error-correction (Arrow). With COGENT, we clustered different isoforms into gene families to generate de novo gene contigs. We performed orthology analysis to identify closely related sequences between our transcriptome and other avian and human gene sets. We also aligned our transcriptome against the Calypte anna genome where possible. Finally, we closely examined homology of critical lipid metabolic genes between our transcriptome data and avian and human genomes. We confirmed high levels of sequence divergence within hummingbird lipogenic enzymes, suggesting a high probability of adaptive divergent function in the hepatic lipogenic pathways. Our results have leveraged cutting-edge technology and a novel bioinformatics pipeline to provide a compelling first direct look at the transcriptome of this incredible organism.

genomics↗