This protocol describes a method for obtaining long-read sequence data from human blood. The result of running this protocol should be an increase in sequencing output per PromethION flow cell with minimal addition of cost. This method can be scaled to process multiple human genomes in parallel. For example, using all 48 positions on a PromthION 48 would generate 48 human genomies at 30 coverage and with a read N50 >20 kb every 72 hours.

This method is as follows:

The protocol starts with 1 ml of human blood, which yields a final library of 1-2 µg DNA. This library is split into 3 aliquots, and each aliquot is sequenced one after the other on one PromethION flow cell. The flow cell is washed using the Flow Cell Wash Kit (EXP-WSH004) before loading the next library aliquot. Internally, this method has yielded an extra 25% of sequencing data compared to sequencing without washing the re-loading.

• 1 ml human blood collected in K2-EDTA
• QIAGEN Puregene Blood Kit
• 2X “size selection buffer” (2.5% w/v PVP 360000, 1.2 M NaCl, 20 mM Tris.HCl pH 8)
• Qubit dsDNA BR Assay Kit (ThermoFisher Scientific)
• Megaruptor 3 Shearing Kit (Diagenode, cat # E07010003)
• Ligation Sequencing Kit V14 (SQK-LSK114)
• Sequencing Auxiliary Vials V14 (EXP-AUX003)
• Flow Cell Wash Kit (EXP-WSH004)
• 80% ethanol in nuclease-free water
• Isopropanol
• TE buffer (1 mM EDTA, pH 8.0)
• 1.5 ml Eppendorf DNA LoBind tubes
• 15 ml Falcon tubes
• Centrifuge and rotor for 15 ml Falcon tubes
• Incubator or water bath (set at 37°C and 50°C)
• Diagenode Megaruptor 3
• Vortex mixer
• Qubit fluorometer

Please note: It is recommended to use wide-bore pipette tips throughout the entire protocol to avoid unwanted shearing.

1. Freeze 1 ml of blood in K2-EDTA at -20°C for 24 hours to lyse the cells.

2. Extract the DNA as described in the Rabbit blood DNA - QIAGEN Puregene Blood Kit.

3. Quantify 1 μl of eluted sample using a Qubit fluorometer and the Qubit dsDNA BR Assay Kit. Take 5-8 μg of DNA into the next step.

4. Follow the Size selection of HMW DNA by semi-selective DNA precipitation protocol to deplete short fragments (<10 kb). Elute the DNA in 65 μl of Elution Buffer (EB) from the Oxford Nanopore Technologies kit. We recommend eluting overnight at room temperature, but if an overnight incubation step is not possible, elute at 60°C for 1 hour.

5. Quantify 1 μl of eluted sample using a Qubit fluorometer and the Qubit dsDNA BR Assay Kit. Take all remaining DNA into the next step.

6. Transfer 65 μl of DNA into the Megaruptor 3 and shear at the desired N50 (we recommend 20-30 kb).

Note: Internal results show that an N50 of 20 kb gives a slightly higher output than an N50 of 30 kb.

7. Quantify 1 μl of eluted sample using a Qubit fluorometer and the Qubit dsDNA BR Assay Kit. Take 48 μl of DNA into the next step.

8. Follow the protocol for gDNA library preparation using the Ligation Sequencing Kit V14 for PromethION, and use the Long Fragment Buffer (LFB) at the Adapter ligation and clean-up step.

Please note: At the end of the Adapter ligation and clean-up step, resuspend the library in 25 μl Elution Buffer (EB) and incubate at 37°C for 10 minutes to improve the recovery of long fragments.

9. Quantify 1 μl of eluted sample using a Qubit fluorometer and the Qubit dsDNA BR Assay Kit. Take all remaining DNA into the next step.

10. Split the library between three 1.5 ml Eppendorf DNA LoBind tubes and make up each library to 32 μl at 10-20 fmol, using Elution Buffer (EB). Store two of the tubes at 2-4°C until ready to use.

11. Take the third tube with 32 μl of library and add 100 μl Sequencing Buffer (SB) and 68 μl Library Beads (LIB). Proceed to priming the PromethION flow cell and loading the library mix. For this step, we recommend using standard pipette tips as wide-bore tips will not fit in the inlet port of the flow cell.

12. When configuring your sequencing run in MinKNOW, select the experiment to run for 72 hours. Use the Advanced options drop-down and ensure the reserve pores toggle is on.

Figure 1. Experiment parameters set-up screen on the MinKNOW software. The run length is configurable by the user, and should be set to 72 hours. In the Advanced options, a toggle can be switch on for pores to be held in reserve for later stages of the sequencing run.

13. After 24-30 hours, pause the sequencing run using the Pause button at the top of the experiment screen in MinKNOW, and wash the flow cell following the Flow Cell Wash Kit protocol.

Figure 2. Sequencing run control panel in the MinKNOW software, with the option to pause a run and flush the flow cell.

14. While the flow cell is incubating with the flow cell wash mix for 60 minutes, prepare a new library for loading using one of the stored tubes with 32 μl of library, and the reagents in the Sequencing Auxiliary Vials V14.

15. Close the inlet port cover, and remove any buffer from the waste port at the top of the flow cell. Add Flush Cell Flush (FCF) and the second library to the flow cell. Resume the sequencing run.

Figure 3. To remove the waste buffer from a PromethION flow cell, the inlet port cover needs to be closed (rotated anti-clockwise). The waste liquid can then be aspirated from either one of the waste ports, labelled 2 and 3 in the image.

16. After 48-54 hours, pause the sequencing run in MinKNOW and repeat the flow cell wash and library reloading in steps 13-15. Resume the sequencing run and continue sequencing until 72 hours have elapsed.

• Yield: 3.5 μg after DNA extraction, size selection and fragmentation
• OD 260/280: 1.88
• OD 260/280: 2.16

Figure 4. Nanodrop trace showing the absorbance for a typical DNA sample after extraction, size selection and fragmentation.

Fragmentation length distribution:

Figure 5. FEMTO Pulse trace of fragment length distributions at each step in the protocol. Size selection and Megaruptor shearing brings the modal fragment length to 40-50 kb.

Libraries were prepared using the Ligation Sequencing Kit:

• Read length profile:

Figure 6. Read length distribution from a typical sequencing experiment using this protocol.

• Cumulative output over time:

Figure 7. Sequencing output from a typical sequencing experiment with two flow cell washes (labelled on the graph).

In conclusion, this protocol opens up the opportunity to scale up sequencing experiments to maximise output without compromising read length and at minimal additional cost. This allows parallel sequencing of multiple human genomes at high coverage. The size selection and fragmentation steps ensure that the DNA fragments are of a consistent length, and output data shows that read N50s of 20-30 kb can be routinely achieved.

Supporting protocols:

• Rabbit blood DNA - QIAGEN Puregene Blood Kit
• Size selection of HMW DNA by semi-selective DNA precipitation
• Ligation Sequencing Kit V14 for PromethION
• Flow Cell Wash Kit (EXP-WSH004)

To see our range of sequencing kits and expansion kits, please visit the Nanopore Store.