This is a WIP, actively being updated.
I have acquired many lab materials and consumables to sequence my own genome at home. It took me about a month to get everything I needed to do a full end to end high quality run. This is pretty dense to read, so I would encourage you filter it through your preferred AI to help step by step, should you wish to follow it.
The costs are out of reach for the average person but they are decreasing and we will eventually have affordable technology, like a cell phone or AI, telling us about our DNA + RNA expression at any given time.
Note: I have the equivalent RNA equipment and materials and will be doing a follow up post on sequencing RNA. You want both- DNA gives you a longer term view of what your cell state is at, but RNA tells you what proteins are actually being expressed at a particular point in time. This doesn’t represent all cells, just the ones that you collect in that section of the body.
Equipment + Materials
Hardware
- Oxford Nanopore Technologies MinION ($7.5k)
- Laptop/workstation for MinKNOW (any PC should be fine)
- 100GB+ storage for outputs ($300)
- 4070ti GPU for Dorado basecalling ($3k)
- Vortex ($50)
- Heat block ($250)
- Centrifuge ($400 used on eBay)
Consumables
- SQK-LSK114 Ligation Sequencing Kit V14 for DNA
- SQK-RNA004 Direct RNA Sequencing Kit
- EXP-WSH004 Flow Cell Wash Kit
- EXP-CTL001 Control material
- PBS 1x
Reagents
- DNA extraction kit
- Monarch HMW DNA Extraction Kit for Cells & Blood
- Nuclei Prep Buffer
- Nuclei Lysis Buffer
- RNase A
- Proteinase K
- Precipitation Enhancer
- DNA Capture Beads
- gDNA Wash Buffer
- Elution Buffer II
- Bead retainers / Monarch collection parts from kit
- DNA library-prep reagents
- NEBNext Companion Module v2 / repair-end prep reagents
- FFPE DNA Repair Buffer
- FFPE DNA Repair Mix
- Ultra II End-prep Reaction Buffer
- Ultra II End-prep Enzyme Mix
- NEBNext Quick T4 DNA Ligase
- AMPure XP beads
- Fresh 80% ethanol
- Nuclease-free water
- Long Fragment Buffer / LFB
- Elution Buffer / EB
- Ligation Adapter / LA
- Ligation Buffer / LNB
- Sequencing Buffer / SB
- Library Beads / LIB
- Flow Cell Flush / FCF
- Flow Cell Tether / FCT
- BSA
- DNA quantity measurement
- Qubit fluorometer
- Qubit dsDNA BR or HS Assay Kit
- Bench equipment
- Microcentrifuge
- Vortex mixer
- Heat block / dry bath
- Magnetic rack for 1.5/2 mL tubes
- Tube racks
- Ice bucket / cold block
- Freezer at -20°C
- Fridge at 4°C
- Pipettes
- P1000
- P200
- P20
- P10
Plastics / Lab Pro-style consumables
This is the plastics list we discussed, including the Lab Pro-type items.
- Sterile flocked cheek swabs
- 1.5 mL microcentrifuge tubes
- 2.0 mL microcentrifuge tubes
- DNA LoBind 1.5 mL tubes
- RNA LoBind tubes
- 0.2 mL PCR tubes
- Qubit assay tubes
- P1000 sterile filtered tips
- P200 sterile filtered tips
- P20 sterile filtered tips
- P10 sterile filtered tips
- Wide-bore P200 tips
- Tube labels / lab marker
- Gloves
Software stack
- MinKNOW
- Dorado
- minimap2
- samtools
- mosdepth
- NanoPlot or pycoQC
- Clair3
- DeepVariant, optional
- Ensembl VEP
- ClinVar
- gnomAD
- PharmGKB
- dbSNP, optional
- Python/R
- SQLite/Postgres for query layer later
End-to-end DNA sequencing protocol
The goal is to go from 2 cheek-swab samples → MinION sequencing
0. Setup
- Put on gloves.
- Wipe bench, pipettes, tube rack, and centrifuge area with 70% ethanol or lab disinfectant.
- Label two 1.5–2 mL tubes:
- Sample A
- Sample B
- Label two more tubes as balance tubes if needed.
- Put PBS on ice or in the fridge.
- Set heat block / thermomixer to 56°C.
- Take Proteinase K and RNase A from 20°C freezer only when preparing the Monarch solutions.
- Keep Nuclei Prep Buffer cold.
- Confirm centrifuge tubes are always balanced directly opposite each other.
1. Collect cheek cells
Sample A
- Rinse mouth with water.
- Wait 10 minutes.
- Do not brush teeth.
- Do not use mouthwash.
- Open sterile swab without touching the swab tip.
- Scrape inside cheek firmly for 60 seconds.
- Add 1 mL cold PBS to the tube labeled Sample A using P1000.
- Put swab head into the PBS.
- Vortex 10 seconds.
- Press swab against tube wall to recover liquid.
- Remove and discard swab.
Sample B
- Use a new sterile swab.
- Scrape inside cheek firmly for 60 seconds.
- Add 1 mL cold PBS to the tube labeled Sample B using P1000.
- Put swab head into the PBS.
- Vortex 10 seconds.
- Press swab against tube wall to recover liquid.
- Remove and discard swab.
2. Pellet cells
- Put Sample A and Sample B opposite each other in the centrifuge.
- Spin at 2,000 × g for 30 seconds.
- Confirm white pellet/material is at the bottom of each tube.
- For Sample A, use P1000 to remove most PBS without touching pellet.
- For Sample A, use P200 to remove more PBS, leaving 50–100 µL above pellet.
- For Sample B, repeat the same PBS removal.
- Gently flick each tube to resuspend the pellet in the remaining liquid.
3. Prepare Monarch lysis solutions for 2 samples
The Monarch manual says to prepare Nuclei Prep Solution by mixing Nuclei Prep Buffer with RNase A, and Nuclei Lysis Solution by mixing Nuclei Lysis Buffer with Proteinase K; for 2 standard-input samples, the table gives 330 µL Nuclei Prep Buffer + 11 µL RNase A and 330 µL Nuclei Lysis Buffer + 22 µL Proteinase K.
Nuclei Prep Solution
- Get a clean tube.
- Label it Nuclei Prep Solution.
- Add 330 µL Nuclei Prep Buffer.
- Add 11 µL RNase A.
- Vortex to mix.
- Keep on ice.
Nuclei Lysis Solution
- Get a clean tube.
- Label it Nuclei Lysis Solution.
- Add 330 µL Nuclei Lysis Buffer.
- Add 22 µL Proteinase K.
- Vortex to mix.
- Keep at room temperature.
- Put RNase A and Proteinase K back at 20°C after use.
4. Lyse cells
For each sample, NEB’s cell workflow uses 150 µL Nuclei Prep Solution, then 150 µL Nuclei Lysis Solution, then 56°C for 10 minutes.
Sample A
- Add 150 µL Nuclei Prep Solution using P200.
- Pipette up/down 10× gently.
- Incubate 2 minutes at room temperature.
- Add 150 µL Nuclei Lysis Solution using P200.
- Invert 10×.
- Do not vortex.
- Put tube in heat block at 56°C for 10 minutes.
Sample B
- Add 150 µL Nuclei Prep Solution using P200.
- Pipette up/down 10× gently.
- Incubate 2 minutes at room temperature.
- Add 150 µL Nuclei Lysis Solution using P200.
- Invert 10×.
- Do not vortex.
- Put tube in heat block at 56°C for 10 minutes.
5. Bind DNA to Monarch beads
For standard-input cells, the Monarch workflow adds 75 µL Precipitation Enhancer, 2 DNA Capture Beads, and 275 µL isopropanol per sample. NEB says to mix on a vertical rotating mixer, or invert gently by hand 30×, with each inversion taking about 5–6 seconds.
Sample A
- Add 75 µL Precipitation Enhancer using P200.
- Invert 8–10×.
- Use clean forceps to add 2 DNA Capture Beads.
- Add 275 µL isopropanol using P1000.
- Close tube.
- Invert slowly by hand 30×.
- Each inversion should take 5–6 seconds.
- Do not vortex.
Sample B
- Add 75 µL Precipitation Enhancer using P200.
- Invert 8–10×.
- Use clean forceps to add 2 DNA Capture Beads.
- Add 275 µL isopropanol using P1000.
- Close tube.
- Invert slowly by hand 30×.
- Each inversion should take 5–6 seconds.
- Do not vortex.
6. Wash DNA-bound beads
NEB specifies 500 µL gDNA Wash Buffer, invert 2–3×, remove wash, then repeat the wash.
Sample A
- Use P1000/P200 to remove liquid without removing beads/DNA.
- Add 500 µL gDNA Wash Buffer using P1000.
- Invert 2–3×.
- Remove wash buffer without losing beads.
- Add another 500 µL gDNA Wash Buffer.
- Invert 2–3×.
- Remove wash buffer.
Sample B
- Use P1000/P200 to remove liquid without removing beads/DNA.
- Add 500 µL gDNA Wash Buffer using P1000.
- Invert 2–3×.
- Remove wash buffer without losing beads.
- Add another 500 µL gDNA Wash Buffer.
- Invert 2–3×.
- Remove wash buffer.
7. Elute DNA
NEB says to pour beads into the bead retainer, pulse spin ≤1 sec, move beads to a fresh Monarch tube, add 100 µL Elution Buffer II, incubate 5 minutes at 56°C, then spin 30 seconds at 12,000 × g to collect eluate.
Sample A
- Put Monarch bead retainer into Monarch spin collection tube.
- Pour beads into bead retainer.
- Close cap.
- Pulse spin ≤1 second to remove leftover wash buffer.
- Remove bead retainer from collection tube.
- Pour beads into a clean Monarch 2 mL tube.
- Add 100 µL Elution Buffer II directly onto beads.
- Incubate 5 minutes at 56°C.
- Put bead retainer into a clean labeled DNA LoBind tube.
- Pour eluate + beads into bead retainer.
- Spin 30 seconds at 12,000 × g.
- Discard beads and bead retainer.
- Keep eluate; this is Sample A purified genomic DNA.
Sample B
- Put Monarch bead retainer into Monarch spin collection tube.
- Pour beads into bead retainer.
- Close cap.
- Pulse spin ≤1 second to remove leftover wash buffer.
- Remove bead retainer from collection tube.
- Pour beads into a clean Monarch 2 mL tube.
- Add 100 µL Elution Buffer II directly onto beads.
- Incubate 5 minutes at 56°C.
- Put bead retainer into a clean labeled DNA LoBind tube.
- Pour eluate + beads into bead retainer.
- Spin 30 seconds at 12,000 × g.
- Discard beads and bead retainer.
- Keep eluate; this is Sample B purified genomic DNA.
8. Homogenize DNA
NEB says HMW DNA must be homogenized before measurement/use; pipette eluate 5–10× with a wide-bore P200 tip, and for rapid homogenization incubate 30–60 minutes at 37°C before pipetting again.
Sample A
- Use a wide-bore P200 tip.
- Pipette eluate up/down 5–10× slowly.
- Incubate at 37°C for 30–60 minutes if the DNA looks viscous/stringy.
- Pipette up/down 5–10× again.
Sample B
- Use a wide-bore P200 tip.
- Pipette eluate up/down 5–10× slowly.
- Incubate at 37°C for 30–60 minutes if the DNA looks viscous/stringy.
- Pipette up/down 5–10× again.
9. Qubit quantify both DNA samples
Thermo’s Qubit dsDNA HS protocol uses a 1:200 reagent:buffer working solution, 190 µL working solution + 10 µL standard, final assay volume 200 µL, and 2 minutes room-temperature incubation before reading.
For 2 samples + 2 standards, make enough working solution for 4 tubes.
- Label Qubit tubes:
- S1
- S2
- Sample A
- Sample B
- Make 800 µL Qubit working solution:
- 796 µL Qubit dsDNA HS Buffer
- 4 µL Qubit dsDNA HS Reagent
- Mix working solution by pipetting or vortexing.
- Add 190 µL working solution to tube S1.
- Add 10 µL Standard #1 to tube S1.
- Vortex 2–3 seconds.
- Add 190 µL working solution to tube S2.
- Add 10 µL Standard #2 to tube S2.
- Vortex 2–3 seconds.
- Add 198 µL working solution to tube Sample A.
- Add 2 µL Sample A DNA.
- Vortex 2–3 seconds.
- Add 198 µL working solution to tube Sample B.
- Add 2 µL Sample B DNA.
- Vortex 2–3 seconds.
- Incubate all tubes 2 minutes at room temperature, protected from strong light.
- On Qubit, select dsDNA HS.
- Read S1.
- Read S2.
- Read Sample A.
- Read Sample B.
- Record concentrations in ng/µL.
- Calculate total DNA:
total DNA ng = concentration ng/µL × remaining volume µL- Choose the better sample for sequencing today.
10. Decide what goes into SQK-LSK114
ONT’s LSK114 DNA protocol calls for 1 µg high-molecular-weight genomic DNA, adjusted to 47 µL with nuclease-free water.
- If Sample A or Sample B has at least 1,000 ng total DNA, use 1,000 ng.
- Calculate volume:
DNA volume µL = 1000 ng / Qubit concentration ng/µL- Put that volume of DNA into a clean 1.5 mL DNA LoBind tube.
- Add nuclease-free water to bring total volume to 47 µL.
- Mix gently by pipetting or flicking.
- Briefly spin down.
- If both samples are below 1,000 ng, use the best sample, but expect lower output.
- Do not combine Sample A + Sample B unless you are okay losing identity or you have a barcoding kit.
11. DNA repair and end-prep
ONT’s LSK114 repair/end-prep reaction is 60 µL total: 47 µL DNA, optional 1 µL DNA Control Sample, 3.5 µL FFPE Repair Buffer, 2 µL FFPE DNA Repair Mix, 3.5 µL Ultra II End-prep Reaction Buffer, and 3 µL Ultra II End-prep Enzyme Mix; incubation is 20°C for 5 min, then 65°C for 5 min.
- Thaw NEB repair/end-prep reagents on ice.
- Flick/invert reagent tubes to mix.
- Spin down tubes before opening.
- Do not vortex enzyme mixes.
- Get a 0.2 mL PCR tube.
- Add 47 µL DNA sample.
- Optional: add 1 µL DNA Control Sample.
- If omitting control, add 1 µL more DNA/water so total remains correct.
- Add 3.5 µL NEBNext FFPE DNA Repair Buffer.
- Add 2 µL NEBNext FFPE DNA Repair Mix.
- Add 3.5 µL Ultra II End-prep Reaction Buffer.
- Add 3 µL Ultra II End-prep Enzyme Mix.
- Total volume should be 60 µL.
- Gently pipette mix 10–20×.
- Briefly spin down.
- Incubate in thermocycler/heat block:
- 20°C for 5 minutes
- 65°C for 5 minutes
- Hold at 4°C or place on ice.
12. Clean repaired DNA with AMPure XP beads
ONT says to add 60 µL AMPure XP Beads to the end-prep reaction, incubate 5 minutes, wash with fresh 80% ethanol, elute in 61 µL nuclease-free water, then retain 61 µL eluate.
- Vortex AMPure XP beads until fully resuspended.
- Transfer the 60 µL end-prep reaction to a clean 1.5 mL DNA LoBind tube.
- Add 60 µL AMPure XP beads.
- Mix by flicking.
- Incubate 5 minutes at room temperature.
- Put tube on magnetic rack.
- Wait until solution is clear/colorless.
- Keep tube on magnet.
- Remove and discard supernatant.
- Add 200 µL freshly prepared 80% ethanol without disturbing beads.
- Remove ethanol.
- Add another 200 µL freshly prepared 80% ethanol.
- Remove ethanol.
- Spin down briefly.
- Put back on magnet.
- Remove residual ethanol with P10/P20.
- Air-dry bead pellet ~30 seconds.
- Do not let pellet crack.
- Remove tube from magnet.
- Add 61 µL nuclease-free water.
- Resuspend beads gently.
- Incubate 2 minutes at room temperature.
- Put tube back on magnet.
- Wait until eluate is clear/colorless.
- Transfer 61 µL eluate to a clean DNA LoBind tube.
13. Adapter ligation
ONT’s LSK114 ligation reaction is 100 µL total: 60 µL DNA, 25 µL Ligation Buffer, 10 µL NEBNext Quick T4 DNA Ligase, and 5 µL Ligation Adapter, incubated 10 minutes at room temperature. ONT specifically recommends using the LNB supplied in the LSK114 kit rather than third-party ligase buffer.
- Thaw Ligation Buffer / LNB at room temperature.
- Mix LNB by pipetting; it is viscous.
- Spin down.
- Place on ice.
- Thaw Elution Buffer / EB.
- Vortex EB.
- Spin down.
- Place on ice.
- Thaw Long Fragment Buffer / LFB.
- Vortex LFB.
- Spin down.
- Place on ice.
- In a clean DNA LoBind tube, add 60 µL repaired/end-prepped DNA.
- Add 25 µL Ligation Buffer / LNB.
- Pipette mix 10–20×.
- Add 10 µL NEBNext Quick T4 DNA Ligase.
- Pipette mix gently.
- Add 5 µL Ligation Adapter / LA.
- Pipette mix gently.
- Total volume should be 100 µL.
- Briefly spin down.
- Incubate 10 minutes at room temperature.
14. Adapter-ligated library cleanup
ONT says to add 40 µL AMPure XP Beads, incubate 5 minutes, wash beads twice with 250 µL Long Fragment Buffer or Short Fragment Buffer, elute in 25 µL Elution Buffer, incubate 10 minutes, and retain 25 µL eluate.
- Vortex AMPure XP beads until fully resuspended.
- Add 40 µL AMPure XP beads to the 100 µL ligation reaction.
- Mix by flicking.
- Incubate 5 minutes at room temperature.
- Put tube on magnetic rack.
- Wait until solution clears.
- Keep tube on magnet.
- Remove and discard supernatant.
- Add 250 µL Long Fragment Buffer / LFB.
- Flick tube to resuspend beads.
- Spin down briefly.
- Put back on magnet.
- Wait until clear.
- Remove and discard supernatant.
- Add another 250 µL Long Fragment Buffer / LFB.
- Flick tube to resuspend beads.
- Spin down briefly.
- Put back on magnet.
- Wait until clear.
- Remove and discard supernatant.
- Spin down briefly.
- Put back on magnet.
- Remove residual buffer with P10/P20.
- Air-dry ~30 seconds.
- Do not let pellet crack.
- Remove tube from magnet.
- Add 25 µL Elution Buffer / EB.
- Resuspend beads gently.
- Incubate 10 minutes at room temperature.
- For HMW DNA, use 37°C incubation if recovery is poor.
- Put tube on magnet.
- Wait at least 1 minute, until eluate is clear/colorless.
- Transfer 25 µL eluate to a clean DNA LoBind tube.
- This is your prepared sequencing library.
- Keep library on ice or at 4°C until loading.
15. Quantify final library
ONT recommends loading 35–50 fmol final library for high-output simplex data, or 10–20 fmol for duplex; if below recommendation, use the whole yield and make to 32 µL EB.
- Use 1 µL of prepared library for Qubit.
- Make Qubit tube:
- 199 µL working solution
- 1 µL library
- Vortex 2–3 seconds.
- Incubate 2 minutes.
- Read Qubit.
- Record library concentration.
- If you can calculate fmol, target 35–50 fmol.
- If not calculating fmol today, use 12 µL library in the loading mix below.
16. Prepare flow cell
ONT says FLO-MIN114 flow cells should sit at room temperature for 20 minutes, LSK114 is compatible with FLO-MIN114, and active pores should be checked before loading.
- Take flow cell out of fridge.
- Leave at room temperature for 20 minutes.
- Plug MinION into computer.
- Open MinKNOW.
- Insert flow cell under clip.
- Press down firmly on priming port cover to ensure contact.
- In MinKNOW, run flow cell check.
- Confirm enough active pores.
- If pore count is terrible, do not waste library unless you accept a failed run.
17. Prime flow cell
ONT’s MinION priming mix per flow cell is 1,170 µL Flow Cell Flush + 5 µL BSA + 30 µL Flow Cell Tether, total 1,205 µL; then load 800 µL via the priming port, wait 5 minutes, then load another 200 µL after opening SpotON.
- Thaw:
- Sequencing Buffer / SB
- Library Beads / LIB
- Flow Cell Tether / FCT
- Flow Cell Flush / FCF
- Vortex thawed reagents.
- Spin down.
- Put on ice.
- In a clean tube, prepare priming mix:
- 1,170 µL Flow Cell Flush / FCF
- 5 µL BSA 50 mg/mL
- 30 µL Flow Cell Tether / FCT
- Mix by pipetting.
- Slide priming port cover clockwise to open.
- Check for air bubble.
- Set P1000 to 200 µL.
- Put tip into priming port.
- Draw back to 220–230 µL to remove 20–30 µL buffer/air.
- Do not expose pore array to air.
- Load 800 µL priming mix into priming port slowly.
- Avoid bubbles.
- Wait 5 minutes.
18. Prepare library loading mix
ONT’s loading mix per MinION flow cell is 37.5 µL Sequencing Buffer, 25.5 µL Library Beads, and 12 µL DNA library, total 75 µL.
- Immediately before use, mix Library Beads / LIB by pipetting.
- In a new DNA LoBind tube, add:
- 37.5 µL Sequencing Buffer / SB
- 25.5 µL Library Beads / LIB
- 12 µL DNA library
- Total volume should be 75 µL.
- Mix gently by pipetting.
- Do not vortex library loading mix.
19. Finish priming and load library
ONT says to gently lift the SpotON cover, load 200 µL priming mix into the priming port, then add 75 µL prepared library through the SpotON sample port dropwise.
- Gently lift SpotON sample port cover.
- Load 200 µL priming mix into the priming port.
- Mix prepared library gently by pipetting.
- Add 75 µL prepared library to the SpotON sample port dropwise.
- Wait for each drop to enter before adding next drop.
- Avoid bubbles.
- Replace SpotON cover.
- Close priming port.
- Put light shield on flow cell.
- Close MinION lid.
20. Start sequencing in MinKNOW
You would have installed MinKNOW by now if you went out and bought your own ONT MinION, but this is where you actually start sequencing. Everything up until now has been prep (crazy, right?). When you start sequencing, you go into MinKNOW, enter experiment/sample details, select SQK-LSK114, configure outputs, and click Start.
- In MinKNOW, click Start sequencing.
- Enter experiment name:
DNA_SampleA_LSK114_YYYYMMDD- Enter sample ID.
- Select kit:
SQK-LSK114- Select flow cell:
FLO-MIN114- Enable basecalling if your machine can handle it.
- Choose Dorado HAC model for first run.
- Output:
- POD5 on
- FASTQ on if basecalling live
- BAM optional
- Set run length:
- 6 hours for test run
- 24–72 hours for more output
- Click Start.
- Watch first 10–20 minutes.
- Confirm pores are sequencing.
- Confirm reads are accumulating.
- Confirm no immediate pore crash.
21. Basecall after run if needed
- Find POD5 output directory.
- Install Dorado.
- Run basecalling:
dorado basecaller sup pod5_directory/ > calls.bam- Convert BAM to FASTQ if needed:
samtools fastq calls.bam > reads.fastq- For faster first pass, use HAC instead of SUP:
dorado basecaller hac pod5_directory/ > calls.bam22. Align reads to human reference
- Download GRCh38 FASTA.
- Index reference:
minimap2-d GRCh38.mmi GRCh38.fa- Align reads:
minimap2-ax map-ont GRCh38.mmi reads.fastq | samtoolssort-o aligned.bam- Index BAM:
samtools index aligned.bam- Check alignment summary:
samtools flagstat aligned.bam > flagstat.txt- Check coverage:
mosdepth sample_cov aligned.bam23. Variant calling
- Install Clair3.
- Use ONT model.
- Run Clair3 with GRCh38 reference, sorted BAM, and output directory.
- Output should include VCF.
- Do not overinterpret low-coverage variants.
- For a first MinION run, treat this as technical validation, not medical-grade interpretation.
24. Annotation
- Install VEP.
- Annotate VCF against GRCh38.
- Add ClinVar.
- Add gnomAD.
- Add PharmGKB later.
- Store final table with columns:
- chromosome
- position
- ref
- alt
- gene
- consequence
- ClinVar significance
- gnomAD frequency
- genotype
- read depth
- variant quality
Results
[TBD]
What can I do with my own genome?
[TBD, may make this its own article]