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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

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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

W Gu et al. Genome Biol.

Abstract

Next-generation sequencing has generated a need for a broadly applicable method to remove unwanted high-abundance species prior to sequencing. We introduce DASH (Depletion of Abundant Sequences by Hybridization). Sequencing libraries are 'DASHed' with recombinant Cas9 protein complexed with a library of guide RNAs targeting unwanted species for cleavage, thus preventing them from consuming sequencing space. We demonstrate a more than 99 % reduction of mitochondrial rRNA in HeLa cells, and enrichment of pathogen sequences in patient samples. We also demonstrate an application of DASH in cancer. This simple method can be adapted for any sample type and increases sequencing yield without additional cost.

Figures

Fig. 1
Fig. 1
a S. pyogenes Cas9 protein binds specifically to DNA targets that match the ‘NGG’ protospacer adjacent motif (PAM) site. Additional sequence specificity is conferred by a single guide RNA (sgRNA) with a 20 nucleotide hybridization domain. DNA double strand cleavage occurs three nucleotides upstream of the PAM site. b Depletion of Abundant Sequences by Hybridization (DASH) is used to target regions that are present at a disproportionately high copy number in a given next-generation sequencing library following tagmentation or flanking sequencing adaptor placement. Only non-targeted regions that have intact adaptors on both ends of the same molecule are subsequently amplified and represented in the final sequencing library
Fig. 2
Fig. 2
Depletion of Abundant Sequences by Hybridization (DASH) targeting abundant mitochondrial ribosomal RNA in HeLa RNA extractions. a Normalized coverage plots showing alignment to the full-length human mitochondrial chromosome. Before treatment, three distinct peaks representing the 12S and 16S ribosomal subunits characteristically account for a large majority of the coverage (>60 % of total mapped reads). After treatment, the peaks are virtually eliminated — with 12S and 16S signatures reduced 1000-fold to 0.055 % of mapped reads. b Coverage plot of Cas9-targeted region with 12S and 16S gene boundaries across the top. Each red arrowhead represents one sgRNA target site. We chose 54 target sites, spaced approximately 50 bp apart. c Scatterplot of the log of fragments per kilobase of transcript per million mapped reads (log-fpkm) values per human gene in the control versus treated samples illustrate the significant reduction in reads mapping to the targeted 12S and 16S genes. DASH treatment results in 82- and 105-fold reductions in coverage for the 12S and 16S subunits, respectively. The slope of the regression line (red) fit to the untargeted genes indicates a 2.38-fold enrichment in reads mapped to untargeted transcripts. R-squared (R 2) value of the regression line (0.979) indicates minimal off-target depletion. Between replicates, the R2 coefficient between fpkm values across all genes is 0.994, indicating high reproducibility (three replicates). Notably, one gene, MT-RNR2-L12 (MT-RNR2-like pseudogene), shows significant depletion in the DASHed samples compared with the control
Fig. 3
Fig. 3
Normalized coverage plots of DASH-treated (orange) and untreated (blue) libraries generated from patient cerebrospinal fluid (CSF) samples with confirmed infections. Targeted mitochondrial rRNA genes (left) and representative genes for pathogen diagnosis (right) are depicted for the following: patient 1, Balamuthia mandrillaris (a), patient 2, Cryptococcus neoformans (b), patient 3, Taenia solium (c). Across all cases, the DASH technique significantly reduced the coverage of human 12S and 16S genes by an average of 7.5-fold while increasing the coverage depth for pathogenic sequences by an average 5.9-fold. See Table 1 for relevant data
Fig. 4
Fig. 4
a DASH is used to selectively deplete one allele while keeping the other intact. An sgRNA in conjunction with Cas9 targets a wild-type (WT) KRAS sequence. However, since the G12D (c.35G>A) mutation disrupts the PAM site, Cas9 does not efficiently cleave the mutant KRAS sequence. Subsequent amplification of all alleles using flanking primers, as in the case of digital PCR, Sanger sequencing, or high-throughput sequencing, is only effective for non-cleaved and mutant sites. b Three human genomic DNA samples with varying ratios of wild-type to mutant (G12D) KRAS were treated either with KRAS-targeted DASH, a non-human control DASH, or no DASH. Counts of intact wild-type and G12D sequences were then measured by droplet digital PCR (ddPCR). c Same data as in (b), presented as percentage of mutant sequences detected. Inset shows fold enrichment of the percentage of mutant sequences with KRAS-targeted DASH versus no DASH. For both (b) and (c), values and error bars are the average and standard deviation, respectively, of three independent experiments

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