Timothy Gilpatrick's research while affiliated with Johns Hopkins University and other places

Publications (12)

Preprint
The mitochondrial genome (mtDNA) is an important source of disease-causing genetic variability, but existing sequencing methods limit understanding, precluding phased measurement of mutations and clear detection of large sporadic deletions. We adapted a method for amplification-free sequence enrichment using Cas9 cleavage to obtain full length nano...
Article
Full-text available
Probing epigenetic features on DNA has tremendous potential to advance our understanding of the phased epigenome. In this study, we use nanopore sequencing to evaluate CpG methylation and chromatin accessibility simultaneously on long strands of DNA by applying GpC methyltransferase to exogenously label open chromatin. We performed nanopore sequenc...
Article
Full-text available
Despite recent improvements in sequencing methods, there remains a need for assays that provide high sequencing depth and comprehensive variant detection. Current methods1–4 are limited by the loss of native modifications, short read length, high input requirements, low yield or long protocols. In the present study, we describe nanopore Cas9-target...
Article
Background: Telomerase reverse transcriptase (TERT) promoter mutations play a role in carcinogenesis and are found in both tumors and cancer cell lines. TERT promoter methylation, transcription factor binding, chromatin remodeling, and alternative splicing are also known to play an integral role in TERT regulation. Methodology: Using nanopore Ca...
Article
Full-text available
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
Article
Full-text available
High-throughput complementary DNA sequencing technologies have 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 modifications are not retained. We address these limitations using a native poly(A) RNA sequencing stra...
Preprint
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...
Preprint
Full-text available
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 reads ¹ . Existing strategies are currently limited by high input DNA requirements, low yield, short (<5kb)...
Article
Although development of high throughput RNA sequencing technologies has allowed significant advances in our understanding of transcriptome complexity and regulation, certain questions remain intractable with conventional short read cDNA sequencing. Native RNA sequencing technology can address these difficult questions; including identity and freque...
Preprint
Full-text available
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 and chromatin accessibility, with long-read nanopore sequencing technology, a method we call nanoNOMe. We...
Preprint
Full-text available
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...

Citations

... Using the haplotype-tagged PromethION reads from "WhatsHap haplotag," we classified the reads into two haplotypes and calculated the methylation frequencies in each haplotype category using the given script in nanopolish. We converted the BAM file by nanopore-methylation-utilities 65 for visualization in the Integrative Genomics Viewer (IGV) 66 (version 2.8.9 and 2.11.9) via the bisulfite mode using nanopolish results. The methylation frequency for the region covered with ≥3 reads was visualized as a heatmap in IGV. ...
... Beyond the regulation by the ETS/MAPK axis, TERT promoter has been shown to be subjected to other regulatory inputs, some of which might be modified by the presence of TPMs, whereas others likely operate as determinants of TERT baseline transcriptional repression. Although this is beyond the scope of this paper, the role of genomic insulator CTCF, which likely determines long-distance interactions [31] as well as the contribution of DNA methylation and allele-specific histone marks, which were recently characterized in thyroid cancer cell lines [18,32,33], are worth noting. Overall, these add to the idea of a tightly regulated, multi-faceted control of TERT transcription, which only becomes unchecked during cancer transformation. ...
... minION sequencing. Long-read sequencing was performed on the II1 T-DOM alleles (II1 T-DOM 320 and II1 T-DOM 542) following the nCATS protocol with minor changes 29 . Yolk sacs were isolated from embryos containing the II1 T-DOM transgene and digested with Tail Digestion Buffer (see above) and Proteinase K overnight at 55°C with no shaking. ...
... Additionally, the application of these sequencing technologies to miRNA editing might require further adaptation. In addition to the mentioned methods, Nanopore sequencing technologies could allow the direct detection of inosines in full-length pri-miRNAs, pre-miRNAs, and intronic miRNAs, overcoming several technical issues that hamper editing detection, including those linked to PCR amplification [108,109]. ...
... (SAMtools view -b -F 2320). The poly(A) tails of reads were assessed using the poly(A) estimation module of the program Nanopolish v.0.13.2 (54). Only reads fulfilling quality criteria (tagged as "PASS") were taken into consideration in further analyses. ...
... For example, identifying pathogenic DNA in a patient lung fluid sample requires bypassing human DNA-which often represents > 99% of the sequences-to find the pathogen sequences. Biochemical methods for target sequence enrichment, such as PCR [2][3][4][5], hybrid capture [6], or CRISPR/Cas9 enrichment [7,8] require much more time, expertise, and equipment. In contrast, a computational approach to enriching target sequences provides clear savings of time, labor, and cost. ...
... Beyond the regulation by the ETS/MAPK axis, TERT promoter has been shown to be subjected to other regulatory inputs, some of which might be modified by the presence of TPMs, whereas others likely operate as determinants of TERT baseline transcriptional repression. Although this is beyond the scope of this paper, the role of genomic insulator CTCF, which likely determines long-distance interactions [31] as well as the contribution of DNA methylation and allele-specific histone marks, which were recently characterized in thyroid cancer cell lines [18,32,33], are worth noting. Overall, these add to the idea of a tightly regulated, multi-faceted control of TERT transcription, which only becomes unchecked during cancer transformation. ...
... Devices from Oxford Nanopore Technologies (ONT) enable sequencing of native DNA and RNA molecules with no theoretical upper limit on read length 1 . This supports the accurate assembly and phasing of repetitive genomes and metagenomes [2][3][4][5][6] ; enhanced detection of structural and/or complex genetic variation [7][8][9][10][11] ; assembly-free detection and quantification of spliced RNA transcripts 12 ; and profiling of diverse epigenetic and RNA modifications [13][14][15][16][17][18] . High-output benchtop ONT instruments (GridION and PromethION) have recently enabled cost-effective nanopore sequencing of human and other large eukaryotic genomes 7,8,19 . ...
... Unlike short-read and long-read cDNA sequencing, longread RNA sequencing, also known as dRNA-seq (DRS), does not require cDNA generation and therefore can eliminate the errors that occur during cDNA amplification and avoid RNA-RNA chimaeras produced by cDNA [58]. Although the limitation of reading length is not the challenge with the technique, the fragmentation of the input read is still challenging [59,60]. ...