Biology subjects

Gilpatrick, T.

Publications and source records attributed to Gilpatrick, T..

5 recordsLinked to original sources

bioRxiv2023

IVT generation of guideRNAs for Cas9-enrichment Nanopore Sequencing

Generating high-coverage sequencing coverage at select genomic loci has extensive applications in both research science and genetic medicine. Long-read sequencing technologies (e.g. nanopore sequencing) have expanded our ability to generate sequencing data in regions (e.g. repetitive elements) that are difficult to interrogate with short-read sequencing methods. In work presented here, we expand on our previous work using CRISPR/Cas9 for targeted nanopore sequencing by using in vitro transcribed guideRNAs, with 1100 guideRNAs in a single experiment. This approach decreases the cost per guideRNA, increases the number of guideRNAs that can be multiplexed in a single experiment, and provides a way to rapidly screen numerous guideRNAs for cutting efficiency. We apply this strategy in multiple patient-derived pancreatic cancer cell lines, demonstrating its ability to unveil structural variation in "deletion hotspots" around the tumor suppressor genes p16 (CDKN2A), and SMAD4.

molecular biology↗

bioRxiv2022

Long read mitochondrial genome sequencing using Cas9-guided adaptor ligation

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 nanopore reads of mtDNA. We then utilized the long reads to phase mutations in a patient with an mtDNA-linked syndrome and demonstrated that this method can map age-induced mtDNA deletions. We believe this method will offer deeper insight into our understanding of mtDNA variation.

genomics↗

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↗

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↗