Jan 06, 2026
Improved Cas9-targeted nanopore sequencing facilitates ultra-deep analysis of genomic variation
- Nick Vereecke1,
- Megan Behrmann2,
- Anupama Khare2,
- John Dekker1
- 1Bacterial Pathogenesis and Antimicrobial Resistance Section (BPARS), Laboratory of Clinical Immunology & Microbiology (LCIM), National Institute for Allergy and Infectious Disease (NIAID), National Institutes of Health (NIH), Bethesda MD;
- 2Laboratory of Molecular Biology (LMB), National Cancer Institute (NCI), National Institutes of Health (NIH), Bethesda MD
Protocol Citation: Nick Vereecke, Megan Behrmann, Anupama Khare, John Dekker 2026. Improved Cas9-targeted nanopore sequencing facilitates ultra-deep analysis of genomic variation. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldrq5ng5b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 15, 2025
Last Modified: January 06, 2026
Protocol Integer ID: 118311
Keywords: R10.4.1, long-read sequencing, Cas9 targeted sequencing, Staphylococcus aureus, Bacteroides, library preparation, antimicrobial resistance, bead capture, heteroresistance, gene amplification, whole genome sequencing, nanopore adapter, nanopore, whole genome, read sequencing, terminal thymine nucleotides of the ont ligation adapter, sequencing method, enriched cas9, separation from background genomic dna, background genomic dna, sequencing efficiency, improved cas9, genomic variation, sequencing yield, dynamic gene amplification, cas9, ligated target, unamplified dna, microbiology, dna, previous cas9, bacteroide, gene, ont ligation adapter, thymine nucleotide
Funders Acknowledgements:
Division of Intramural Research of the National Institute of Allergy
Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research
Abstract
Here we present nanopore adapter-enriched Cas9-targeted sequencing (nAECATS), a method permitting inexpensive, ultra-deep, selective long-read sequencing of targeted regions in native, unamplified DNA. To develop this approach, we modified previous Cas9-targeted sequencing methods, adding a novel bead-based capture step that utilizes the 5’ stretch of eight terminal thymine nucleotides of the ONT ligation adapter for additional purification. This poly(T) region distinguishes the Cas9-enriched adapter-ligated target from non-ligated material, allowing highly specific enrichment and separation from background genomic DNA. Following extensive optimization, we tested the nAECATS protocol on a fixed 10 kb region of interest (ROI) within the Bacteroides fragilis genome and achieved 90% on-target sequencing yield with a mean target coverage of 51,000x from a single Flongle flow cell (353-fold increase in mean ROI coverage versus whole genome sequencing). We next applied nAECATS to a more challenging target of variable length (>20 kb) in a Staphylococcus aureus strain containing a dynamic gene amplification previously demonstrated to confer resistance to the fluoroquinolone antibiotic delafloxacin. Absolute ROI coverage of 46,000x and 74% on-target sequencing yield were achieved with a MinION flow cell, revealing individual S. aureus populations with 2-4 tandem amplifications at the single cell level, and demonstrating the potential of this approach to reveal novel underlying biology. While loss of sequencing efficiency and potential shearing were observed with progressively longer fragments (up to 41 kb), significant improvements in enrichment were still achieved. We anticipate that nAECATS ultra-deep sequencing will find broad application for a wide range of biological questions in pro- and eukaryotic (epi)genomics and microbiology.
Protocol materials
streptavidin magnetic beads New England BiolabsCatalog #S1420S
rCutSmart BufferNew England BiolabsCatalog #B6004S
Alt-R® CRISPR-Cas9 tracrRNAIDT TechnologiesCatalog #1072532
Nuclease Free Duplex BufferIDT TechnologiesCatalog #11-01-03-01
Alt-R® S.p. HiFi Cas9 Nuclease V3IDTCatalog #1081060
Quick CIPNew England BiolabsCatalog #M0525S
dATP Solution (100 mM)New England BiolabsCatalog #N0440S
Taq DNA Polymerase with Standard Taq BufferNew England BiolabsCatalog #M0273S
Qubit dsDNA BR Assay kitInvitrogen - Thermo FisherCatalog #Q32850
Genomic DNA ScreenTape AnalysisAgilent TechnologiesCatalog #5067-5365
Quick Ligation Kit - 150 reactionsNew England BiolabsCatalog #M2200L
Maxwell® HT 96 gDNA Blood Isolation SystemPromegaCatalog #A2670
Nanobind CBB kitPacBioCatalog #102-301-900
1.5mL DNA LoBind tubesEppendorfCatalog #0030108051
AMPure XP Beads (AXP)Beckman CoulterCatalog #AXP
Qubit dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q32851
Ligation Sequencing Kit V14Oxford Nanopore TechnologiesCatalog #SQK-LSK114
Troubleshooting
Preparation of High Molecular Weight (HMW) gDNA
2h
For the preparation of bacterial HMW gDNA we refer to either of following kits and associated extraction protocols.
Note - care should be taken to always follow good practices to prevent shearing of HMW gDNA (e.g., excessive mixing, vortexing, and the use of small pipetting volumes)
Note - always check that the composition of the elution buffer does not contain EDTA as a simple Tris-HCl elution buffer is preferred for ONT long-read downstream applications
Note - HMW gDNA can be stored at 4 °C for several weeks. Fresh material is recommended and any freezing/thawing should be avoided for long-read sequencing purposes.
Note - Always check the quality and quantity of the HMW gDNA using different methods, including NanoDrop QC, Qubit quantification, and TapeStation QC.
2h
Maxwell® HT 96 gDNA Blood Isolation SystemPromegaCatalog #A2670
Nanobind CBB kitPacBioCatalog #102-301-900
Proceed to Cas9 target enrichment - Preparing the Cas9 ribonucleoprotein complexes (RNPs)
Cas9 target enrichment - Preparing the Cas9 ribonucleoprotein complexes (RNPs)
1h 7m 10s
Thaw an aliquot of rCutSmart BufferNew England BiolabsCatalog #B6004S , mix by vortexing, spin down in a microfuge to collect any liquid in the bottom of the tube, and place on ice.
5m
Thaw the crRNA probes and Alt-R® CRISPR-Cas9 tracrRNAIDT TechnologiesCatalog #1072532 on ice, mix by vortexing, spin down in a microfuge to collect any liquid in the bottom of the tube, and place on ice.
Pool the crRNA probes for each cleavage reaction in a 1.5mL DNA LoBind tubesEppendorfCatalog #0030108051 tube by combining equal volumes of each crRNA probe (100 micromolar (µM) in TE (7.5 )).
Note - for a double-flanked target, pool 1 µL of each crRNA into a 0.2 mL thin-walled PCR tube single crRNA or many crRNA probes (up to ~100) may be used in a single cleavage reaction.
Note - unused crRNA probe mix may be stored at -80 °C and minimal freeze thaw recommended (e.g., aliquots of 1 µL )
5m
Anneal the pooled crRNAs with tracrRNA in Nuclease Free Duplex BufferIDT TechnologiesCatalog #11-01-03-01 by assembling the following in a 0.2 ml thin-walled PCR tube, as follows:
| Reagent | Volume | |
| nuclease-free Duplex Buffer (IDT) | 8 μL | |
| crRNA pool (100 μM, equimolar; see above) | 1 μL | |
| tracrRNA (100 μM) | 1 μL | |
| Total | 10 μL |
5m
Mix well by pipetting and spin down in a microfuge to collect any liquid in the bottom of the tube.
1m
Start the thermal cycler protocol (95 °C for 00:05:10 ) and only place the tubes in the thermal cycler when the first 00:00:10 have passed.
10s
Incubate the crRNA:tracrRNA reaction mix at 95 °C for 00:05:00 , but only place the tubes in the thermal cycler when 95 °C has been reached.
10m
Remove the tube from the thermal cycler and allow it to cool to room temperature and spin down to collect any liquid in the bottom of the tube.
Note - Do not use a thermal cycler for this step.
Note - Storage and reuse of the annealed mix is not recommended.
5m
Spin down the Alt-R® S.p. HiFi Cas9 Nuclease V3IDTCatalog #1081060 in a microfuge and place on a freezer block (-20 °C ).
30s
To form Cas9 RNPs, assemble the following components in a 1.5 ml Eppendorf DNA LoBind tube in the following order:
| Reagent | Volume | |
| Nuclease-free water | 79.2 μL | |
| Annealed crRNA:tracrRNA mix (10 μM) | 10 μL | |
| NEB rCutSmart Buffer (10X) | 10 μL | |
| HiFi Cas9 (62 μM) | 0.8 μL | |
| Total | 100 μL |
5m
Mix well by flicking the tube and spin down in a microfuge to collect any liquid in the bottom of the tube.
30s
Form the RNPs by incubating the tube at 21 °C for 00:30:00 in a thermal cycler (lid heating off), then return the RNPs on ice until required.
Note - Proceed to the he next step (Dephosphorylating genomic DNA) during the 00:30:00 RNP incubation step
30m
Cas9 target enrichment - Dephosphorylating genomic DNA
46m 30s
Thaw the Quick CIPNew England BiolabsCatalog #M0525S , mix the Quick CIP in the tube by pipetting up and down, and keep at room temperature.
5m
Prepare the HMW gDNA in nuclease-free water by transferring 5 µg HMW gDNA into a 0.2 ml thin-walled PCR tubes.
Note - Adjust to 24 µL with nuclease-free water if highly concentrated.
5m
Mix thoroughly by flicking the tube avoiding unwanted shearing and spin down in a microfuge to collect any liquid in the bottom of the tube.
30s
Assemble the following components in a clean 0.2 ml thin-walled PCR tube:
| Reagent | Volume | |
| NEB rCutsmart buffer (10X) | 3 μL | |
| HMW gDNA (at >210 ng/μL) | 24 μL | |
| Total | 27 μL |
5m
Ensure the components are thoroughly mixed by pipetting five times using a P200 pipet, and spin down in a microfuge to collect any liquid in the bottom of the tube.
30s
Add 3 µL of Quick CIP to the tube, mix gently by flicking the tube, and spin down in a microfuge to collect any liquid in the bottom of the tube.
30s
Using a thermal cycler, incubate at 37 °C for 00:20:00 , followed by 80 °C for 00:04:00 and hold at 21 °C (room temperature).
30m
Cas9 target enrichment - Cleaving and dA-tailing target DNA
1h 8m 30s
Thaw the dATP Solution (100 mM)New England BiolabsCatalog #N0440S tube, vortex to mix thoroughly, and place on ice.
5m
Spin the Taq DNA Polymerase with Standard Taq BufferNew England BiolabsCatalog #M0273S down in a microfuge and place on a freezer block (-20 °C ).
30s
Prepare a 10 millimolar (mM) dATP solution in a fresh 1.5 ml DNA LoBind tube by adding 2 µL of the 100 millimolar (mM) dATP solution to 18 µL of nuclease-free water, vortex to mix, spin down in a minifuge to collect any liquid in the bottom of the tube, and keep on ice.
2m
Add the following to the 0.2 mL thin-walled PCR tubes, containing 30 μl dephosphorylated DNA sample:
| Reagent | Volume | |
| Dephosphorylated HMW gDNA | 30 μL | |
| Cas9 RNPs | 10 μL | |
| 10 mM dATP | 1 μL | |
| NEB Taq polymerase | 1 μL | |
| Total | 42 μL |
5m
Carefully mix the contents of the tube by gentle inversion, spin down in a microfuge to collect any liquid in the bottom of the tube, and place the tube in the thermal cycler.
1m
Using the thermal cycler, incubate at 37 °C for 00:20:00 , followed by 72 °C for 00:10:00 , and hold at 4 °C or return the tube on ice.
Note - The Cas9 enzyme is active at 37 °C and denatured at 72 °C .
Note - A 15 minute (range between 10-60 minutes) cut time is recommended by default. Longer 37ºC incubations may increase the amount of off-target reads without increasing the yield of on-target reads, while shorter incubations may result in incomplete target cleavage. However, some regions may benefit from a longer incubation at 37 °C .
35m
Perform quality and quantity control on 1 µL of the Cas9 enriched product using Qubit dsDNA BR Assay kitInvitrogen - Thermo FisherCatalog #Q32850 and Genomic DNA ScreenTape AnalysisAgilent TechnologiesCatalog #5067-5365 .
20m
Keep samples on ice and proceed to Preparation of buffers for Poly(A) enrichment and Long-read Ligation Library preparation (SQK-LSK114).
Preparation of buffers for Poly(A) enrichment
20m
Prepare Wash/Bind buffer (20 millimolar (mM) Tris-HCl, 0.5 Molarity (M) NaCl, 1 millimolar (mM) EDTA @ 7.5 ) by adding:
| Reagent | Volume | |
| nuclease-free water | 4.6 mL | |
| NaCl (1M) | 5 mL | |
| Tris-HCl (1M, pH 7.5) | 0.2 mL | |
| EDTA (0.05M, pH 8.0) | 0.2 mL | |
| Total | 10 mL |
5m
Prepare 2X Wash/Bind buffer (40 millimolar (mM) Tris-HCl, 1 Molarity (M) NaCl, 2 millimolar (mM) EDTA @ 7.5 ) by adding:
| Reagent | Volume | |
| NaCl (1M) | 9.2 mL | |
| Tris-HCl (1M, pH 7.5) | 0.4 mL | |
| EDTA (0.05M, pH 8.0) | 0.4 mL | |
| Total | 10 mL |
5m
Prepare Low-Salt buffer (20 millimolar (mM) Tris-HCl, 0.15 Molarity (M) NaCl, 1 millimolar (mM) EDTA @ 7.5 ) by adding:
| Reagent | Volume | |
| nuclease-free water | 8.1 mL | |
| NaCl (1M) | 1.5 mL | |
| Tris-HCl (1M, pH 7.5) | 0.2 mL | |
| EDTA (0.05M, pH 8.0) | 0.2 mL | |
| Total | 10 mL |
5m
Long-read Ligation library preparation (SQK-LSK114)
34m
Thaw Ligation Buffer (LNB; part of SQK-LSK114) and Quick T4 Ligase Buffer from the Quick Ligation Kit - 150 reactionsNew England BiolabsCatalog #M2200L at room temperature, mix by pipetting, spin down in a microfuge to collect any liquid in the bottom of the tube, and keep on ice.
5m
Thaw Elution Buffer (EB; part of SQK-LSK114) at room temperature, mix by vortexing, spin down in a microfuge to collect any liquid in the bottom of the tube, and keep on ice.
5m
Thaw Long Fragment Buffer (LFB; part of SQK-LSK114) at room temperature, mix by vortexing, spin down in a microfuge to collect any liquid in the bottom of the tube, and keep on ice.
5m
Compose the following ligation reaction based on the number of Poly(A)35 you would like to pool:
Note - When 10 Poly(A) RXN are pooled, split the reaction into 2x5 Ligation RXN for convenience.
| Reagent | 1 Poly(A) RXN | 3 Poly(A) RXN | 5 Poly(A) RXN | |
| Poly(A) enriched Cas9 sample | 42 µL | 126 µL | 210 µL | |
| Ligation Buffer (LNB) | 20 µL | 30 µL | 50 µL | |
| Quick T4 Ligase Buffer | NA | 30 µL | 50 µL | |
| Quick T4 Ligase | 10 µL | 30 µL | 50 µL | |
| Ligation Adapter (LA) | 5 µL | 5 µL | 5 µL | |
| Nuclease-free Water | NA | 10 µL | 20 µL | |
| Total | 77 µL | 231 µL | 385 µL |
3m
Mix by flicking the tube and spin down in a microfuge to collect any liquid in the bottom of the tube.
30s
Incubate the Ligation reaction for 00:15:00 at room temperature on a Hula mixer.
15m 30s
Ligation Adapter (LA) enrichment using Poly(A) paramagnetic beads
2h 21m 30s
Vortex the streptavidin magnetic beads New England BiolabsCatalog #S1420S well to homogenize them.
30s
Transfer 125 µL beads (= 500 µg beads) into a 1.5 mL LoBind Tube.
30s
Add 100 µL Wash/Binding Buffer (20 millimolar (mM) Tris-HCl, 0.5 Molarity (M) NaCl, 1 millimolar (mM) EDTA @ 7.5 ), vortex to resuspend the beads, and spin down in a microfuge to collect any liquid in the bottom of the tube.
30s
Place the tube on a magnetic rack and leave for 00:00:30 to pellet beads
1m
Remove supernatant and discard.
1m
Add 100 µL Wash/Binding Buffer, vortex to resuspend the beads, and spin down in a microfuge to collect any liquid in the bottom of the tube.
30s
Place the tube on a magnetic rack and leave for 00:00:30 to pellet beads.
1m
Remove supernatant and discard.
30s
Repeat step 44-46 TWICE with fresh Wash/Binding Buffer.
5m
Thaw the TEG-biotinylated poly(A)35 (at 8 pmol/µL in 20 millimolar (mM) Tris-HCl,0.5 Molarity (M) NaCl, 1 millimolar (mM) EDTA @ 7.5 ) on ice, mix by vortexing, spin down in a microfuge to collect any liquid in the bottom of the tube, and place on ice.
Add 25 µL of TEG-biotinylated poly(A)35 (at 8 pmol/µL in 20 millimolar (mM) Tris-HCl, 0.5 Molarity (M) NaCl, 1 millimolar (mM) EDTA @ 7.5 ) and vortex to resuspend the beads and oligos.
30s
Incubate at Room Temperature for 00:30:00 on a Hula Mixer.
30m
Place the tube on a magnetic rack and leave for 00:00:30 to pellet beads.
1m
Remove supernatant and keep in a fresh 1.5 mL Eppendorf DNA LoBind tube.
Note - this will allow to assess how much biotinylated bait is left using the Qubit ssDNA kit.
30s
Wash beads by adding 100 µL Wash/Binding Buffer, vortex to resuspend the beads, and spin down in a microfuge to collect any liquid in the bottom of the tube.
30s
Place the tube on a magnetic rack and leave for 00:00:30 to pellet beads.
1m
Remove supernatant and discard.
30s
Repeat Step 53-55 TWICE with fresh Wash/Binding Buffer.
4m
Add 77 µL /231 µL /385 µL 2X Wash/Binding buffer (40 millimolar (mM) Tris-HCl, 1 Molarity (M) NaCl, 2 millimolar (mM) EDTA @ 7.5 ) to the magnetic beads mixture, flick the tube to resuspend the beads, and spin down in a microfuge to collect any liquid in the bottom of the tube.
Note - Volume depends on number of pooled reactions in the Long-read Ligation library preparation (SQK-LSK114) procedure
30s
Add 77 µL /231 µL /385 µL of the Cas9 enriched and adapter ligated DNA sample (i.e., ~5 µg in 50 µL EB (part of SQK-LSK114)) to the magnetic beads mixture, flick the tube to resuspend the beads, and spin down in a microfuge to collect any liquid in the bottom of the tube.
Note - Volume depends on number of pooled reactions in the Long-read Ligation library preparation (SQK-LSK114) procedure
30s
Incubate at Room Temperature for 00:30:00 on a Hula Mixer.
30m
Place the tube on a magnetic rack and leave for 00:02:00 to pellet beads.
2m 30s
Remove supernatant and keep in a fresh 1.5 mL Eppendorf DNA LoBind tube.
Note - this will only serve as control for QC using Qubit dsDNA BR and TapeStation gDNA ScreenTape.
30s
Wash beads by adding 100 µL Wash/Binding Buffer, flick the tube to resuspend the beads, and spin down in a microfuge to collect any liquid in the bottom of the tube.
30s
Place the tube on a magnetic rack and leave for 00:02:00 to pellet bead.
2m 30s
Remove supernatant and discard.
30s
Repeat Step 62-64 for a second wash with fresh Wash/Binding Buffer.
2m
Add 100 µL cold Low Salt Buffer (20 millimolar (mM) Tris-HCl, 0.15 Molarity (M) NaCl, 1 millimolar (mM) EDTA @ 7.5 ; kept on ice) to the magnetic beads mixture, flick the tube to resuspend the beads, and spin down in a microfuge to collect any liquid in the bottom of the tube.
30s
Immediately place the tube on a magnetic rack and leave for 00:00:30 to pellet beads.
1m
Immediately remove supernatant and discard.
30s
Immediately add 20 µL pre-warmed (37 °C ) Elution Buffer (10 millimolar (mM) Tris-HCl @ 7.5 ) to the magnetic beads mixture, flick the tube to resuspend the beads, and spin down in a microfuge to collect any liquid in the bottom of the tube.
Note - Volume can be changed depending on downstream application.
30s
Incubate for 00:30:00 at 37 °C on a Hula Mixer.
30m
Place the tube on a magnetic rack and leave for 00:02:00 to pellet beads.
2m 30s
Transfer supernatant to a fresh 1.5 mL Eppendorf DNA LoBind Tube and keep on ice.
Note – This is the fraction of our interest.
30s
Optional - Repeat Step 69-72 for a second eluate. Though this generally does not result in extra recovery.
3m
Perform quality and quantity control on 1 µL of the resulting supernatants and retained fractions (See above) using Qubit dsDNA BR/HS and TapeStation gDNA ScreenTape.
Note - For optimal recovery and sequencing efficiency it is recommended to omit quality and quantity control on the eluate to optimize input for sequencing.
15m
Proceed immediately to Adapter removal using AMPure XP Beads.
Adapter removal using AMPure XP Beads
1h 5m 30s
Allow the AMPure XP Beads (AXP)Beckman CoulterCatalog #AXP to get to room temperature.
15m
Vortex the AXP Beads to homogenize them.
30s
Add 8 µL AXP Beads to the Poly(A) enriched Cas9 sample, mix by flicking the tube, and spin down in a microfuge to collect any liquid in the bottom of the tube.
Note - This is a 0.4X clean-up if a 20 µL elution was done in the previous step. Whenever volumes in the previous procedure are changed, change them here as well to guarantee optimal adapter removal.
1m
Incubate on a Hula Mixer for 00:05:00 at room temperature.
5m 30s
Spin the sample down in a microfuge to collect any liquid in the bottom of the tube and pellet on a magnet.
30s
Keep the tube on the magnet for 00:02:00 (or until clear and colourless) and remove supernatant.
2m 30s
Add 250 µL LFB to the tube and gently rotate the 1.5 mL Eppendorf LoBind tube 180º and allow the beads to migrate through the LFB. Once migrated, turn the tube another 180º and allow the beads to migrate through the LFB for a second time.
Note - For some sample types it might be required to resuspend the beads by flicking rather than having them migrate through the LFB. In this case resuspend the beads and allow to pellet against the magnet for 2 minutes.
2m
Keep the tube on the magnet for 00:02:00 (or until clear and colourless) and remove supernatant.
Note - Some samples require flicking of the tube
2m 30s
Repeat Step 82-83 with fresh LFB.
5m
Spin the tube down in a microfuge to collect any liquid in the bottom of the tube, place back on the magnet, and remove residual LFB buffer using a P20 tip.
30s
Allow the tube to dry (lid open) for 00:00:30 .
Note - Do not dry the pellet to the point it shows cracks.
1m
Remove the tube from the magnetic rack, add 7 µL Elution Buffer (EB) onto the pellet, and resuspend by flicking the tube.
Note - For MinION/GridION flow cell library preparation a volume of 12 µL is desirable here as 1 µL is required for the final library loading.
1m
Spin down in a microfuge to collect any liquid in the bottom of the tube and incubate for 00:10:00 at 37 °C on a Hula mixer.
10m 30s
Place the tube on the magnetic rack and pellet the beads for 00:02:00 (or until clear and colourless).
2m 30s
Retain the eluted adapter-depleted Poly(A) enriched Cas9 products and transfer into a fresh 1.5 mL Eppendorf DNA LoBind tube.
30s
Perform quality and quantity control on 1 µL of the adapter-depleted Poly(A) enriched Cas9 product using Qubit dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q32851 and TapeStation gDNA ScreenTape.
Note - For optimal recovery and sequencing efficiency it is recommended to omit quality and quantity control on the eluate to optimize input for sequencing.
15m
Immediately proceed to Long-read Sequencing on Flongle.
Long-read Sequencing on Flongle
10m
For the sequencing on Flongle please refer to the respective protocols for Flongle and MinION/GridION flow cell library prepration and loading as part of the Ligation Sequencing Kit V14Oxford Nanopore TechnologiesCatalog #SQK-LSK114 - Link
10m