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Targeted nanopore sequencing with Cas9-guided adapter ligation


Despite recent improvements in sequencing methods, there remains a need for assays that provide high sequencing depth and comprehensive variant detection. Current methods1,2,3,4 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-targeted sequencing (nCATS), an enrichment strategy that uses targeted cleavage of chromosomal DNA with Cas9 to ligate adapters for nanopore sequencing. We show that nCATS can simultaneously assess haplotype-resolved single-nucleotide variants, structural variations and CpG methylation. We apply nCATS to four cell lines, to a cell-line-derived xenograft, and to normal and paired tumor/normal primary human breast tissue. Median sequencing coverage was 675× using a MinION flow cell and 34× using the smaller Flongle flow cell. The nCATS sequencing requires only ~3 μg of genomic DNA and can target a large number of loci in a single reaction. The method will facilitate the use of long-read sequencing in research and in the clinic.

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Fig. 1: Method schematic and coverage data.
Fig. 2: SNVs.
Fig. 3: Methylation.
Fig. 4: Structural variation.

Data availability

Sequencing data from all non-primary patient samples for this study can be retrieved from the SRA, under the BioProject ID PRJNA531320.

Code availability

The computational code used in all of the analysis is hosted on GitHub (see,


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This work was supported by funding from the National Institutes for Health (grant no. R01 HG009190) (National Human Genome Research Institute).

Author information




T.G. and W.T. constructed the study. T.G. performed the experiments. T.G., I.L. and F.S. analyzed the data. T.G., J.G., E.R., R.B. and A.H. developed the method. S.S. and B.D. provided primary breast tissue and generated the mouse xenografts. T.G. and W.T. wrote the paper.

Corresponding author

Correspondence to Winston Timp.

Ethics declarations

Competing interests

J.G., E.R., R.B. and A.H. are employees of Oxford Nanopore Technologies. W.T. has two patents licensed to Oxford Nanopore Technologies (US patent nos. 8,748,091 and 8,394,584). T.G., I.L., F.S. and W.T. have received travel funds to speak at symposia organized by Oxford Nanopore Technologies.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Integrated supplementary information

Supplementary Fig. 1 Enrichment data at example off-target locus.

(a) Coverage and reads at off-target site (first locus from Supplementary Table 3), identified in sequencing run TG_09. (b) Pair-wise alignment showing similarity between guideRNA and the off-target cleavage site.

Supplementary Fig. 2 True positive variants and false positive variants demonstrating the impetus for dual-strand filter.

Left: Two real variants which are supported by data on both strands. Right: Example of two false positive variants resulting from a sequencing error on only one strand.

Supplementary Fig. 3 Persisting false positive variant that passes dual-strand filter.

The single false positive variant from high-coverage sequencing data that passes dual-strand filtering. This variant is present in a highly thymidine-dense region. Note this variant falls within a repetitive region of the genome masked by RepeatMasker, thus the lowercase reference.

Supplementary Fig. 4 Two other sites in tumor tissue demonstrating loss of heterozygosity on chr17.

Single nucleotide high-confidence variant calls (nanopolish passing dual strand filter) at two other enriched sites on chr17 (KRT19 and 30kb piece of BRCA1). Reads were phased to show only variants passing dual-strand filter using the ‘phase-reads’ module of nanopolish. Tumor reads were phased into haplotypes using only variants from the corresponding normal sample.

Supplementary Fig. 5 Methylation line plots, read-level plots and per-CpG plots for five loci in GM12878 enrichment data.

(a) Line and dot plot of methylation calls made by bismark (WGBS Illumina data: GEO: GSE86765) and nanopolish (Cas9-targeted nanopore data) at all CpGs in the targeted regions. Gene models plotted below for orientation. (b) Read-level methylation plots for five loci in GM12878. (c) Per CpG scatter plot comparing methylation calls made by bismark (WGBS Illumina data: GEO: GSE86765) and nanopolish (Cas9-targeted nanopore data) at all CpGs in the targeted regions. r=0.81 across all 5 sites.

Supplementary Fig. 6

Read-level methylation plots for captured loci in breast cell lines (MCF-10-A, MDA-MB-231, MCF-7).

Supplementary Fig. 7 RNA-seq data for 5 genes in breast cell lines.

Normalized expression data (read counts) for three breast cell lines from existing RNA-seq data (GEO: GSE75168).

Supplementary Fig. 8 Read-level methylation plots for captured loci in primary breast tissue.

Read-level methylation plots for 5 captured loci in fresh breast tissue (reduction mammoplasty, cell-line-derived xenograft, paired tumor/normal). Tumor/normal samples are segregated into haplotypes using only variants from the normal sample.

Supplementary Fig. 9 Chromosome 5 deletion in breast cell lines.

Reads at a small (< 10kb) common structural variant on chromosome 5 from breast cell line nanopore enrichment data (deletion at chromosome 7 is included as main Fig. 4a).

Supplementary Fig. 10 Methylation at heterozygous deletions in MDA-MB-231 breast cell line.

Comparing methylation patterns at heterozygous deletions on chromosome 5 and chromosome 7 in MDA-MB-231 cell line data.

Supplementary Fig. 11 Per-allele coverage plots for large heterozygous deletions in GM12878.

Coverage plots around two large heterozygous deletions in GM12878 (RunID: TG_07). Yellow triangles show points of Cas9 cleavage. Blue lines show coverage of reads assigned to paternal haplotype and red lines show coverage of reads assigned to maternal haplotype. (In both cases, the distance between cuts on the deleted allele is ~10kb and distance between cuts on non-deleted allele is ~80kb).

Supplementary Fig. 12 Per-allele coverage plots at loci without deletions.

Comparing paternal and maternal coverage at two sites in GM12878 using a single cut each side (RunID: TG_01) at sites with no heterozygous SVs between guideRNAs. Unlike at the sites of large heterozygous deletions, we do not see a dramatic bias towards either parental allele.

Supplementary Fig. 13 Reads at the BRCA1 locus for GM12878.

Left: Reads from BRCA1 enrichment with DNA extracted using the Masterpure kit (Lucigen, Cat#MC85200) Right: Reads from BRCA1 enrichment run with DNA extracted using the Nanobind kit (Circulomics, Cat#NB-900-001-0).

Supplementary Fig. 14 Comparison of BRCA1 nanopore reads to PacBio reads at unannotated indels.

(a) Showing whole genome PacBio data around BRCA1 in GM12878 from publicly available data (SRA: SRR9001768 - SRR9001773) (b) Comparison of the three not annotated heterozygous indels found in GM12878 between Cas9-nanopore enrichment data (top) and whole-genome PacBio data (bottom).

Supplementary Fig. 15 Allele-specific BRCA1 methylation analysis in GM12878.

Methylation analysis using nanopolish on each of the two alleles of BRCA1 in GM12878. Reads from enrichment run using Circulomics CBB Nanobind kit for DNA preparation shown.

Supplementary information

Supplementary Figs

Supplementary Figs. 1–15.

Reporting Summary

Supplementary Table 1

The gRNA sequences and details for each sequencing run. Sheet 1: gRNA sequences and target sites for the targeted regions for methylation, SV and SNV interrogation. Sheet 2: details of flow cell, sequencer, sample and gRNAs used in each sequencing run.

Supplementary Table 2

Coverage table for all sites across each of the sequencing runs. Read count, average coverage and on-target percentage for the 10 enrichment sites across sequencing runs.

Supplementary Table 3

Off-target analysis with SURVIVOR: off-target analysis for the GM12878 sequencing run using multiple gRNAs (RunID: TG_09), using the bincov tool from SURVIVOR (Jeffares, D. C. et al. Nat. Commun. 8, 14061 (2017)). On-target loci are colored orange. Maximum coverage shows the highest coverage reached in the specific locus.

Supplementary Table 4

SNV calls with different coverage in GM12878: sensitivity/TPR and F1 score of SNVs detected by different tools at different coverage levels in the enriched 140 kb from GM12878 (RunID: TG_09); 174 annotated SNVs exist in these regions. Analysis limited to SNVs, through comparison with the platinum genome dataset in GM12878. TPR, true positive rate (sensitivity). F1 score is the harmonic mean of precision and recall.

Supplementary Table 5

SNVs called in MDA-MB-231 MinION data (three loci). Sheet 1: SNVs in the MDA-MB-231 cell line identified anew using Nanopolish from nanopore enrichment data at three loci (TP53, BRAF, KRAS). Sheet 2: Nanopolish variants from sheet 1 passing dual-strand filter (high-confidence MDA-MB-231 variants).

Supplementary Table 6

Sniffles calls SVs in three breast cell lines: Sniffles SV calls from enrichment data in the three breast cancer cell lines. For both deletions the ploidy was called as heterozygous (het) in MDA-MB231 and homozygous (homo) in MCF-7.

Supplementary Table 7

Sniffles calls of large SVs in GM12878. Left: reference calls from LongRanger 2.1 analysis of 10x Genomics data from the GIAB consortium. Right: Sniffles SV calls in GM12878. het, heterozygousGT*; homo, homozygous. Note the settings of Sniffles were adjusted to ensure that the genotypes of large deletions in GM12878 were correctly called (see Methods)

Supplementary Table 8

Indels in GM12878 BRCA1 enrichment data. Sheet 1: all indels called between assemblies of the BRCA1 haplotypes in GM12878. DNA isolated using the Circulomics Nanobind CBB kit (RunID: TG_08). Sheet 2: indels from sheet 1 filtered for length ≥3 nt, removing indels resulting from differences in homopolymer length. Indels not previously annotated are colored blue. Comparison with annotated variants from the platinum genomes 2017 hybrid dataset for Hg38 human assembly (Eberle et al. Genome Res. 27(1), 157–164 (2017)).

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Gilpatrick, T., Lee, I., Graham, J.E. et al. Targeted nanopore sequencing with Cas9-guided adapter ligation. Nat Biotechnol 38, 433–438 (2020).

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