CaBagE: a Cas9-based Background Elimation Strategy for Targeted, Long-read DNA Sequencing
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PROVIDER: PRJNA706072 | ENA |
REPOSITORIES: ENA
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Project description:CaBagE: a Cas9-based Background Elimation Strategy for Targeted, Long-read DNA Sequencing
| PRJNA706071 | ENA
Project description:A substantial fraction of the human genome is difficult to interrogate with short-read DNA sequencing technologies due to paralogy, complex haplotype structures, or tandem repeats. Long-read sequencing technologies, such as Oxford Nanopore's MinION, enable direct measurement of complex loci without introducing many of the biases inherent to short-read methods, though they suffer from relatively lower throughput. This limitation has motivated recent efforts to develop amplification-free strategies to target and enrich loci of interest for subsequent sequencing with long reads. Here, we present CaBagE, a method for target enrichment that is efficient and useful for sequencing large, structurally complex targets. The CaBagE method leverages the stable binding of Cas9 to its DNA target to protect desired fragments from digestion with exonuclease. Enriched DNA fragments are then sequenced with Oxford Nanopore's MinION long-read sequencing technology. Enrichment with CaBagE resulted in a median of 116X coverage (range 39-416) of target loci when tested on five genomic targets ranging from 4-20kb in length using healthy donor DNA. Four cancer gene targets were enriched in a single reaction and multiplexed on a single MinION flow cell. We further demonstrate the utility of CaBagE in two ALS patients with C9orf72 short tandem repeat expansions to produce genotype estimates commensurate with genotypes derived from repeat-primed PCR for each individual. With CaBagE there is a physical enrichment of on-target DNA in a given sample prior to sequencing. This feature allows adaptability across sequencing platforms and potential use as an enrichment strategy for applications beyond sequencing. CaBagE is a rapid enrichment method that can illuminate regions of the 'hidden genome' underlying human disease.
| S-EPMC8031414 | biostudies-literature
Project description:Cas9-targeted long-read sequencing of cancer biomarkers
| PRJEB33258 | ENA
Project description:Detection of revertant mosaicism in epidermolysis bullosa through Cas9-targeted long read sequencing
| PRJNA771681 | ENA
Project description:We used the nanopore Cas9 targeted sequencing (nCATS) strategy to specifically sequence 125 L1HS-containing loci in parallel and measure their DNA methylation levels using nanopore long-read sequencing. Each targeted locus is sequenced at high coverage (~45X) with unambiguously mapped reads spanning the entire L1 element, as well as its flanking sequences over several kilobases. The genome-wide profile of L1 methylation was also assessed by bs-ATLAS-seq in the same cell lines (E-MTAB-10895).
2023-09-22 | E-MTAB-12247 | biostudies-arrayexpress
Project description:Integration of the HIV-1 provirus in the host genome ensures a persistent supply of latently infected cells. This latent reservoir is recalcitrant to antiretroviral therapy (ART) making lifelong treatment the only option for patients. The â??shock and killâ?? strategy aims to eradicate latent HIV by reactivating proviral gene expression followed by ART treatment. Gene specific transcriptional activation can be achieved using the RNA-guided CRISPR-Cas9 system comprising small guide RNAs (sgRNAs) with a nuclease deficient Cas9 mutant (dCas9) fused to the VP64 transactivation domain (dCas9-VP64).  We engineered this system to target 23 sites within the LTR promoter of HIV-1 and identified a â??hotspotâ?? for activation. We studied the functionality of activating sgRNAs to transcriptionally modulate the latent proviral genome across multiple different in vitro latency cell models including several J-Lat, ACH2 J1.1 and the CEM T cell model comprising a single clonal population of integrated mCherry-IRES-Tat from a full-length HIV LTR (LChIT). While we observed variable responses of latent cell models to well-characterized chemical stimuli, we detected consistent efficient activation of latent virus mediated by activator sgRNAs. In addition, transcriptome analysis of chemically treated cells revealed massive non-specific gene dysregulation whereas by comparison, dCas9-VP64/sgRNAs induced specific activation of the integrated provirus. In conclusion, we show the potential for CRISPR-mediated gene activation systems to provide enhanced efficiency and specificity in a targeted latency reactivation strategy. This represents a promising approach to a â??functional cureâ?? of HIV/AIDS. Three experimental conditions (sgRNA control, TNF treated and sgRNA against the LTR of HIV-1) were analyzed in triplicate using two sequencing lanes
2016-08-20 | E-GEOD-72259 | biostudies-arrayexpress
Project description:Revertant mosaicism (RM) is a phenomenon in which inherited mutations are spontaneously corrected in somatic cells. RM occurs in some congenital skin diseases, although genetic validation of RM in clinically revertant skin has been challenging, especially when homologous recombination (HR) is responsible for RM. We introduced Nanopore Cas9-targeted sequencing (nCATS) for identifying HR in clinically revertant skin.
2022-01-13 | GSE186002 | GEO
Project description:This dataset contains Xdrop followed by oxford nanopore long read sequencing performed in target tRNA gene deletion (t8) and intergenic region deletion (i50) clones in HepG2 . By applying de novo assembly based approach to Xdrop-LRS data, we identified Cas9-induced on-target genomic alteration.
2022-09-16 | E-MTAB-11327 | biostudies-arrayexpress
Project description:A major challenge for RNA-seq analysis of gene expression is to achieve sufficient coverage of informative non-ribosomal transcripts. In eukaryotic samples, this is typically achieved by selective oligo(dT)-priming of messenger RNAs to exclude ribosomal RNA (rRNA) during cDNA synthesis. However, this strategy is not compatible with prokaryotes in which functional transcripts are generally not polyadenylated. To overcome this, we adopted DASH (Depletion of Abundant Sequences by Hybridization), initially developed for eukaryotic cells, to improve both the sensitivity and depth of bacterial RNA-seq. DASH employs the Cas9 nuclease to remove unwanted cDNA sequences prior to library amplification. We report the design, evaluation, and optimization of DASH experiments for standard bacterial short-read sequencing approaches, including software for automated guide RNA (gRNA) design for Cas9-mediated cleavage in bacterial rDNA sequences. Using these gRNA pools, we effectively removed rRNA reads (56-86%) in RNA-seq libraries from two different model bacteria, the Gram-negative pathogen Salmonella enterica and the anaerobic gut commensal Bacteroides thetaiotaomicron. DASH works robustly, even with sub-nanogram amounts of input cDNA. Its efficiency, high sensitivity, ease of implementation, and low cost (~$5 per sample) render DASH an attractive alternative to rRNA removal protocols, in particular for material-constrained studies where conventional ribodepletion techniques fail.
2020-05-11 | GSE147155 | GEO