Depletion of Abundant Sequences by Hybridization (DASH): using Cas9 to remove unwanted high-abundance species in sequencing libraries and molecular counting applications

Gu, Wei; Crawford, Emily; O’Donovan, Brian; Wilson, Michael R.; Chow, Eric D.; Retallack, Hanna; DeRisi, Joseph L.

doi:10.1186/s13059-016-0904-5

Cited by 292 publications

(303 citation statements)

References 56 publications

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“…Current techniques (Zheng et al, 2016) already allow RNA to be collected from ~50,000 cells in ~10 min, and our GBCs enable higher loading through computational removal of cell doublets. Cost per cell will decline as technologies mature, and sequencing costs can be mitigated through amplification of select targets (like our guide-mapping amplicons) or depletion of uninteresting high abundance genes (Gu et al, 2016). A more subtle point is that intermolecular provirus recombination during transduction can scramble barcode identities in pooled lentivirus preparations (Sack et al, 2016).…”

Section: Discussionmentioning

confidence: 99%

A Multiplexed Single-Cell CRISPR Screening Platform Enables Systematic Dissection of the Unfolded Protein Response

Adamson

Norman

Jost

et al. 2016

Cell

939

947

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SUMMARY Functional genomics efforts face tradeoffs between number of perturbations examined and complexity of phenotypes measured. We bridge this gap with Perturb-seq, which combines droplet-based single-cell RNA-seq with a strategy for barcoding CRISPR-mediated perturbations, allowing many perturbations to be profiled in pooled format. We applied Perturb-seq to dissect the mammalian unfolded protein response (UPR) using single and combinatorial CRISPR perturbations. Two genome-scale CRISPR interference (CRISPRi) screens identified genes whose repression perturbs ER homeostasis. Subjecting ~100 hits to Perturb-seq enabled high-precision functional clustering of genes. Single-cell analyses decoupled the three UPR branches, revealed bifurcated UPR branch activation among cells subject to the same perturbation, and uncovered differential activation of the branches across hits, including an isolated feedback loop between the translocon and IRE1α. These studies provide insight into how the three sensors of ER homeostasis monitor distinct types of stress and highlight the ability of Perturb-seq to dissect complex cellular responses.

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Section: Discussionmentioning

confidence: 99%

A Multiplexed Single-Cell CRISPR Screening Platform Enables Systematic Dissection of the Unfolded Protein Response

Adamson

Norman

Jost

et al. 2016

Cell

939

947

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“…Previous enzymatic approaches for removing WT DNA (19–23) relied on enzymes that digest only at specific sequences, limiting their broad application. The non-selective, DNAse-like activity of DSN in combination with ∼10–15 bp target regions defined by overlapping probes engender NaME-PrO with the potential for massively parallel degradation of WT DNA.…”

Section: Discussionmentioning

confidence: 99%

“…For example in liquid biopsy of cancer using circulating-free DNA (cfDNA) (6–10), traces of somatic mutations serve as biomarkers for early detection (10) or tumor response to treatment (11,12), yet the high excess of circulating WT DNA often dims the opportunities for diagnosis and treatment. While several methods for reducing WT DNA before, during or after polymerase chain reaction (PCR) amplification have been reported (13–18), these usually apply to single or limited numbers of PCR amplicons or are only applicable to a minority of DNA targets recognized by sequence-specific enzymes (19–23). Additionally, PCR amplification itself may contribute to false positive calls in view of polymerase mis-incorporations that are indistinguishable from DNA alterations (24).…”

Section: Introductionmentioning

confidence: 99%

Elimination of unaltered DNA in mixed clinical samples via nuclease-assisted minor-allele enrichment

Song¹,

Liu²,

Fontana³

et al. 2016

Nucleic Acids Res

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Presence of excess unaltered, wild-type (WT) DNA providing no information of biological or clinical value often masks rare alterations containing diagnostic or therapeutic clues in cancer, prenatal diagnosis, infectious diseases or organ transplantation. With the surge of high-throughput technologies there is a growing demand for removing unaltered DNA over large pools-of-sequences. Here we present nuclease-assisted minor-allele enrichment with probe-overlap (NaME-PrO), a single-step approach with broad genome coverage that can remove WT-DNA from numerous sequences simultaneously, prior to genomic analysis. NaME-PrO employs a double-strand-DNA-specific nuclease and overlapping oligonucleotide-probes interrogating WT-DNA targets and guiding nuclease digestion to these sites. Mutation-containing DNA creates probe-DNA mismatches that inhibit digestion, thus subsequent DNA-amplification magnifies DNA-alterations at all selected targets. We demonstrate several-hundred-fold mutation enrichment in diverse human samples on multiple clinically relevant targets including tumor samples and circulating DNA in 50-plex reactions. Enrichment enables routine mutation detection at 0.01% abundance while by adjusting conditions it is possible to sequence mutations down to 0.00003% abundance, or to scan tumor-suppressor genes for rare mutations. NaME-PrO introduces a simple and highly parallel process to remove un-informative DNA sequences and unmask clinically and biologically useful alterations.

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“…In order to reduce the amount of wasted sequencing reads, targeted cleavage of DNA fragments has recently been used to deplete mitochondrial ribosomal RNA-derived fragments in RNA-sequencing libraries 2 . In another study, Wu et al .…”

Section: Introductionmentioning

confidence: 99%

Reducing mitochondrial reads in ATAC-seq using CRISPR/Cas9

Montefiori

Hernandez

Zhang

et al. 2017

Sci Rep

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ATAC-seq is a high-throughput sequencing technique that identifies open chromatin. Depending on the cell type, ATAC-seq samples may contain ~20–80% of mitochondrial sequencing reads. As the regions of open chromatin of interest are usually located in the nuclear genome, mitochondrial reads are typically discarded from the analysis. We tested two approaches to decrease wasted sequencing in ATAC-seq libraries generated from lymphoblastoid cell lines: targeted cleavage of mitochondrial DNA fragments using CRISPR technology and removal of detergent from the cell lysis buffer. We analyzed the effects of these treatments on the number of usable (unique, non-mitochondrial) reads and the number and quality of peaks called, including peaks identified in enhancers and transcription start sites. Both treatments resulted in considerable reduction of mitochondrial reads (1.7 and 3-fold, respectively). The removal of detergent, however, resulted in increased background and fewer peaks. The highest number of peaks and highest quality data was obtained by preparing samples with the original ATAC-seq protocol (using detergent) and treating them with CRISPR. This strategy reduced the amount of sequencing required to call a high number of peaks, which could lead to cost reduction when performing ATAC-seq on large numbers of samples and in cell types that contain a large amount of mitochondria.

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Depletion of Abundant Sequences by Hybridization (DASH): using Cas9 to remove unwanted high-abundance species in sequencing libraries and molecular counting applications

Cited by 292 publications

References 56 publications

A Multiplexed Single-Cell CRISPR Screening Platform Enables Systematic Dissection of the Unfolded Protein Response

A Multiplexed Single-Cell CRISPR Screening Platform Enables Systematic Dissection of the Unfolded Protein Response

Elimination of unaltered DNA in mixed clinical samples via nuclease-assisted minor-allele enrichment

Reducing mitochondrial reads in ATAC-seq using CRISPR/Cas9

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