How to sequence your own DNA at home

# How to sequence your own DNA at home Source: [https://bradleywoolf.com/links-1/sequencing-my-own-dna-at-home](https://bradleywoolf.com/links-1/sequencing-my-own-dna-at-home) I have now sequenced my own genome 5 times with an[Oxford Nanopore Technologies MinION](https://nanoporetech.com/products/sequence/minion)\. This means collecting them from a swab, prepping them for sequencing, running them through a sequencer, then doing analysis over them\. Cheek cells are easily accessible and replenish pretty quickly\. They are not used for cancer diagnosis, inflammation, or what genes are being activated in other parts of the body \(like if you have hives on your chest and want to test what genes are being expressed in the cells that are inflamed\), since you would want to collect the cells having problems and compare them against other normal versions of those cells\. To sequence the cells, I bought lab materials and consumables to sequence my own genome at home\. It took me about two months to get everything together to do a full end to end high quality run\. Likewise,*the costs are still out of reach for the average person*but they are decreasing \(exponentially\!\) and we will eventually have affordable technology, like a cell phone or AI, telling us about our DNA \+ RNA expression real\-time\. ## What can I even do with my own genome? Before we actually spend all this time and money on sequencing, what can we*actually do*with our genome? The genome is not magic by itself\- it is the reference layer\. Once I have a VCF, I can run it through tools like[VEP](https://www.ensembl.org/Tools/VEP),[ClinVar](https://www.ncbi.nlm.nih.gov/clinvar/),[gnomAD](https://gnomad.broadinstitute.org/),[PharmGKB](https://www.clinpgx.org/)\(highly recommend\),[Gene Inspector](https://gene-inspector.pro/), or Claude, and start asking: - Which variants do I have? - Which genes and pathways are affected? - Which medicines might I metabolize differently? - What rare variants should I take seriously? - Where does the model know nothing yet? This last part matters\- the information produced is not yet diagnosis\-level, and it is definitely not “edit yourself with CRISPR because an AI said so\.” The near\-term value is turning a static genome into something queryable, but the “edit yourself with CRISPR” will most likely follow\. DNA is the stable reference, RNA is the current state, and we will eventually integrate all biosensor data into one ‘model’ of yourself\. Here are a list of links and Twitter posts to help: 1. Genetic variants from first principles: 2. Pass your genome \+ RNA to any of these models:[https://www\.biotender\.online/bio\-model\-install\-guide/](https://www.biotender.online/bio-model-install-guide/) 3. Give your genome to Claude Code\- message me if you want me to set this up for you 4. [Take it to a doctor](https://x.com/parmita/status/2024197362286231640)if you metabolize particular drugs differently 5. [Patrick Collison post](https://x.com/patrickc/status/2045164908912968060?lang=en)on using agents to talk to his genome ## Protocol Steps This is intended to be read by AI\- please just copy and paste the URL of this and have ChatGPT walk you through it\. If you have AR glasses, even better, since the AI can walk you through the whole protocol\. ### Hardware - [Oxford Nanopore Technologies MinION](https://store.nanoporetech.com/us/minion.html)\($7\.5k\) - Laptop/workstation for MinKNOW \(any PC should be fine\) - 100GB\+ storage for outputs - GPU for Dorado basecalling - [Vortex](https://www.premiumvials.com/intllab-lab-vortex-mixer-touch-function-lab-vortexer-tattoo-ink-gel-polish-eyelash-adhesives-acylic-paints-test-tubes-and-centrifuge-tubes/?setCurrencyId=1&sku=B08CR43XR3&gad_source=1&gad_campaignid=17182097677&gbraid=0AAAAADpaBkD8X3NHGME9qLpyX764qirew&gclid=Cj0KCQjwk_bPBhDXARIsACiq8R3NH7o4Itb-exPNJG6bpxF2Tcq-r-6r5qv467VZoP-dd-I64DOogK8aAoOqEALw_wcB)\($50\) - [Heat block](https://www.amazon.com/ONiLAB-Mini-Incubator-Aluminium-Calibration/dp/B09VDHS8Z1/ref=sr_1_4?c=ts&dib=eyJ2IjoiMSJ9.DxmMKmWh0Bx0gedZkoko5BEpl9GgEYm-mnndXVtgU_vkjNyUdZb3EOpONsMCc8lEeqeTd62z1zdZ6PLqyHIPJ84nGwhwAJTdaNwja0ciEjuGokdrHcpSKrlrH9LQuEERzNR9d9yjZVuzVIQSTad_id9dKyLBjVrfu_R9Tbexb7CFjdiY8amEbtcmW0EvB8JQpCujFEEuCymGw9aMbx6IBxWmoLCVGHvzHxsNqZOvldQXvblZc19u5Fg3LAog-F6vY6mO063kTaDoGYqLt2lFpL_fiFEs8qZeFawmWniN59E.KheuyTowfnORjWIMj8Z3GE8ZbxOJ3mvUPw2mzbgUolI&dib_tag=se&keywords=Lab%2BHeat%2BBlocks&qid=1779942886&s=industrial&sr=1-4&ts_id=317999011&th=1)\($250\) - [Centrifuge](https://www.ebay.com/p/1401686604?iid=257405680491)\($400 used on eBay\) ### Consumables - [SQK\-LSK114 Ligation Sequencing Kit V14 for DNA](https://store.nanoporetech.com/us/ligation-sequencing-kit-v14.html) - [EXP\-WSH004 Flow Cell Wash Kit](https://store.nanoporetech.com/us/flow-cell-wash-kit-xl.html) - [EXP\-CTL001 Control material](https://store.nanoporetech.com/us/control-expansion.html) - [PBS 1x](https://www.amazon.com/Phosphate-Buffered-Laboratory-Solution-Dilution/dp/B0F37JVYPM/ref=pd_lpo_d_sccl_1/141-3646762-1592425?pd_rd_w=Vio7Q&content-id=amzn1.sym.4c8c52db-06f8-4e42-8e56-912796f2ea6c&pf_rd_p=4c8c52db-06f8-4e42-8e56-912796f2ea6c&pf_rd_r=1RAYMT518CPA4P35Y3JR&pd_rd_wg=8y9gl&pd_rd_r=214b114f-b8e3-4f2a-b195-854c7ee5af81&pd_rd_i=B0F37JVYPM&psc=1) - [Isohelix Buccal swabs](https://isohelix.com/isohelix-dna-rna-buccal-swabs/) ### Reagents - DNA extraction kit - DNA library\-prep reagents - DNA quantity measurement ### Bench equipment 1. Microcentrifuge 2. Vortex mixer 3. Heat block / dry bath 4. Magnetic rack for 1\.5/2 mL tubes 5. Tube racks 6. Ice bucket / cold block 7. Freezer at \-20°C 8. Fridge at 4°C 9. Pipettes ## Plastics / Lab Pro\-style consumables 1. Sterile flocked cheek swabs 2. 1\.5 mL microcentrifuge tubes 3. 2\.0 mL microcentrifuge tubes 4. DNA LoBind 1\.5 mL tubes 5. RNA LoBind tubes 6. 0\.2 mL PCR tubes 7. Qubit assay tubes 8. Pipette sterile filtered tips 9. Wide\-bore P200 tips 10. Tube labels / lab marker 11. [Gloves](https://labproinc.com/products/labclean-unicare-soft-blue-textured-lc-g-nb200-l) ## Software stack 1. MinKNOW 2. Dorado 3. minimap2 4. samtools 5. mosdepth 6. NanoPlot or pycoQC 7. Clair3 8. DeepVariant, optional 9. Ensembl VEP 10. ClinVar 11. gnomAD 12. PharmGKB 13. dbSNP, optional 14. Python/R 15. 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 1. Gloves on\. 2. Clean bench\. 3. Label tubes: 4. Bring AMPure XP beads to room temperature\. 5. Keep enzyme mixes cold\. 6. Set heat block to 56°C\. 7. Keep Nuclei Prep Buffer cold\. 8. Confirm gDNA Wash Buffer already has ethanol added\. 9. Confirm you have isopropanol for DNA binding\. 10. Confirm you have fresh 80% ethanol for AMPure cleanup\. 11. Confirm you have the corrected ONT reagents: - FFPE DNA Repair Buffer v2 - FFPE DNA Repair Mix - N\-Prep Enzyme Mix - Salt\-T4 DNA Ligase - LNB - LA - LFB - EB - SB - LIB - FCF - FCT ## 1\. Collect cheek cells Goal: get as much cheek\-cell material as possible into PBS\. 1. Rinse mouth with water\. 2. Wait 10 minutes\. 3. Do not brush teeth\. 4. Do not use mouthwash\. 5. Scrape inside cheek firmly for 60 seconds\. 6. Add 1 mL cold PBS to labeled tube\. 7. Put swab head into PBS\. 8. Vortex 10 seconds\. 9. Press swab against tube wall to squeeze liquid out\. 10. Remove and discard swab\. What good looks like: ``` PBS may look slightly cloudy. ``` ## 2\. Pellet cheek cells Goal: concentrate cells and remove excess PBS\. 1. Spin at 2,000 × g for 30 seconds\. 2. Look for a small white/off\-white pellet or smear\. 3. Remove most PBS with P1000\. 4. Remove more PBS carefully with P200\. 5. Leave 50–100 µL above the pellet\. 6. Flick gently to resuspend the pellet\. Do not aspirate the pellet\. ## 3\. Prepare Monarch lysis solutions for one sample ## Nuclei Prep Solution ``` 165 µL Nuclei Prep Buffer 5.5 µL RNase A ``` Mix gently\. Keep cold\. You will use 150 µL\. ## Nuclei Lysis Solution ``` 165 µL Nuclei Lysis Buffer 11 µL Proteinase K ``` Mix gently\. Keep at room temperature\. You will use 150 µL\. The extra volume is intentional so pipetting error does not leave you short\. ## 4\. Lyse cells Goal: break open cells and digest proteins while preserving long genomic DNA\. 1. Add 150 µL Nuclei Prep Solution to the resuspended cheek\-cell pellet\. 2. Pipette up/down gently 10 times\. 3. Incubate 2 minutes at room temperature\. 4. Add 150 µL Nuclei Lysis Solution\. 5. Invert tube gently 10 times\. 6. Do not vortex\. 7. Incubate at 56°C for 10 minutes\. What good looks like: ``` The liquid may become more viscous. ``` Do not vortex after lysis\. At this point, the priority is preserving high molecular weight DNA\. ## 5\. Bind DNA to Monarch capture beads Goal: precipitate genomic DNA onto the large Monarch capture beads\. 1. Add 75 µL Precipitation Enhancer\. 2. Invert gently 8–10 times\. 3. Add 2 DNA Capture Beads\. 4. Add 275 µL isopropanol\. 5. Invert slowly 30 times\. 6. Do not vortex\. Important: ``` The beads now carry the DNA. Do not lose the beads. Do not use ethanol here. ``` ## 6\. Wash DNA\-bound beads Goal: wash contaminants away while keeping DNA bound to the capture beads\. 1. Let beads settle briefly\. 2. Remove liquid carefully without removing beads\. 3. Add 500 µL gDNA Wash Buffer\. 4. Invert gently 2–3 times\. 5. Remove wash buffer carefully\. 6. Add another 500 µL gDNA Wash Buffer\. 7. Invert gently 2–3 times\. 8. Remove wash buffer carefully\. 9. Remove as much residual wash as practical without touching or removing beads\. Critical: ``` gDNA Wash Buffer must already have ethanol added. ``` ## 7\. Elute genomic DNA Goal: release purified genomic DNA from the Monarch capture beads\. 1. Put bead retainer into Monarch collection tube\. 2. Transfer/pour beads into the retainer\. 3. Pulse spin ≤1 second\. 4. Move beads to a clean Monarch tube\. 5. Add 100 µL Elution Buffer II\. 6. Incubate at 56°C for 5 minutes\. 7. Put bead retainer over a clean DNA LoBind tube labeled`Extracted DNA`\. 8. Transfer eluate \+ beads into bead retainer\. 9. Spin at 12,000 × g for 30 seconds\. 10. Keep the eluate\. The eluate is the purified genomic DNA\. Do not discard the eluate\. ## 8\. Measure quantity of DNA with a Fluorometer Goal: measure whether there is enough DNA to justify ONT library prep\. Use: ``` 1x dsDNA High Sensitivity ``` Do not select the older non\-1X dsDNA HS workflow if using the premixed 1X reagent\.  ## Standards Standard \#1: ``` 190 µL 1X dsDNA HS working solution 10 µL Standard #1 ``` Standard \#2: ``` 190 µL 1X dsDNA HS working solution 10 µL Standard #2 ``` ## Sample, first attempt ``` 198 µL 1X dsDNA HS working solution 2 µL DNA ``` On Qubit, enter: ## If too low Use more DNA in a fresh Qubit tube: ``` 190 µL 1X dsDNA HS working solution 10 µL DNA ``` On Qubit, enter: Do not just add 8 µL to the old 198 \+ 2 tube, because that makes 208 µL total and breaks the assay math\. Record: ``` Genomic DNA concentration: Remaining DNA volume: Estimated total DNA: ``` Calculate: ``` total DNA ng = Qubit concentration ng/µL × remaining volume µL ```  ## Pause point This is the best place to pause\. For a same\-day break: ``` Store extracted DNA at 4°C / fridge. ``` Before storing: 1. Make sure the DNA is in a clearly labeled DNA LoBind tube\. 2. Quick spin\. 3. Do not vortex\. 4. Close tube tightly\. 5. Put it in the fridge\. Resume at Step 10\. ## 9\. Prepare DNA input for repair/end\-prep Goal: prepare 47 µL DNA input\. Ideal target: Calculate: ``` DNA volume needed = 1000 ng / Qubit concentration ng/µL ``` Then: ``` water volume = 47 µL - DNA volume ``` If the DNA is too dilute and 1,000 ng cannot fit into 47 µL, use the maximum possible volume: ``` 47 µL extracted DNA 0 µL water ``` For the first low\-input run: ``` 0.296 ng/µL × 47 µL = ~13.9 ng DNA ``` That was far below the recommended input, but useful as an end\-to\-end practice run\. ## 10\. Repair / end\-prep Goal: repair DNA and prepare the ends for adapter ligation\. Use the actual v2 reagents: ``` FFPE DNA Repair Buffer v2 FFPE DNA Repair Mix N-Prep Enzyme Mix / Ultra II End Prep Enzyme Mix ``` Do not use Salt\-T4 DNA Ligase in this step\. It is used later\. If not using DCS, replace the optional 1 µL DCS with 1 µL nuclease\-free water\. ## Reaction ``` 47 µL DNA 1 µL nuclease-free water 7 µL FFPE DNA Repair Buffer v2 2 µL FFPE DNA Repair Mix 3 µL N-Prep Enzyme Mix Total = 60 µL ``` ## Steps 1. Add 47 µL DNA to a 0\.2 mL thin\-walled PCR tube labeled`End\-prep`\. 2. Add 1 µL nuclease\-free water\. 3. Add 7 µL FFPE DNA Repair Buffer v2\. 4. Pipette mix 10–20 times\. 5. Add 2 µL FFPE DNA Repair Mix\. 6. Pipette mix 10–20 times\. 7. Add 3 µL N\-Prep Enzyme Mix\. 8. Pipette mix 10–20 times\. 9. Quick spin\. Incubate: ``` 20°C for 5 minutes 65°C for 5 minutes Then place on ice ``` ## 11\. AMPure cleanup after repair/end\-prep Goal: clean the repaired/end\-prepped DNA\. Input: ``` 60 µL repair/end-prep reaction ``` 1. Resuspend AMPure XP beads until uniformly brown\. 2. Add 60 µL AMPure XP beads to the 60 µL reaction\. 3. Mix gently by pipetting 10 times\. 4. Incubate 5 minutes at room temperature\. 5. Put tube on magnet\. 6. Wait until solution clears\. 7. Remove and discard supernatant without touching beads\. ## Wash 1 1. Keep tube on magnet\. 2. Add 200 µL fresh 80% ethanol\. 3. Remove ethanol\. ## Wash 2 1. Add another 200 µL fresh 80% ethanol\. 2. Remove ethanol\. ## Dry 1. Remove residual ethanol with P10/P20\. 2. Air dry briefly, about 30 seconds\. 3. Do not overdry\. 4. Do not let beads crack\. ## Elute 1. Remove tube from magnet\. 2. Add 61 µL nuclease\-free water\. 3. Resuspend beads gently\. 4. Incubate 5–10 minutes at room temperature\. 5. Put tube back on magnet\. 6. Wait until clear\. 7. Transfer 60 µL clear eluate to a clean DNA LoBind tube labeled`Ligation`\. Do not transfer beads\. ## 12\. Adapter ligation Goal: attach ONT sequencing adapters\. Use the actual reagents: ``` LNB LA Salt-T4 DNA Ligase ```  ## Reaction ``` 60 µL repaired/end-prepped DNA 25 µL LNB 10 µL Salt-T4 DNA Ligase 5 µL LA Total = 100 µL ``` ## Steps 1. Use a clean DNA LoBind tube labeled`Ligation`\. 2. Mix LNB slowly by pipetting before adding\. It is viscous\. 3. Add 60 µL repaired/end\-prepped DNA\. 4. Add 25 µL LNB\. 5. Add 10 µL Salt\-T4 DNA Ligase\. 6. Add 5 µL LA\. 7. Mix gently by pipetting 10–15 times\. 8. Do not vortex\. 9. Quick spin\. 10. Incubate 10 minutes at room temperature\. Before incubation, say/check: ``` DNA, LNB, Salt-T4 DNA Ligase, LA. ``` Failure points: ``` Forgetting LA = no sequenceable library. Forgetting Salt-T4 DNA Ligase = adapter ligation fails. Poorly mixed LNB = bad ligation chemistry. Vortexing = unnecessary DNA shearing. ``` ## 13\. Adapter\-ligated library cleanup Goal: remove free adapters, ligase, salts, and small fragments while keeping adapter\-ligated DNA\. Important: ``` This cleanup uses LFB, not ethanol. ``` Input: 1. Resuspend AMPure XP beads fully\. 2. Add 40 µL AMPure XP beads to the 100 µL ligation reaction\. 3. Mix gently by pipetting 10 times\. 4. Incubate 5 minutes at room temperature\. 5. Place tube on magnet\. 6. Wait until solution clears\. 7. Keep tube on magnet\. 8. Remove and discard supernatant carefully\. 9. Do not touch bead pellet\.  ## Wash 1 1. Add 250 µL LFB to beads\. 2. Remove tube from magnet\. 3. Gently resuspend beads by flicking or slow pipetting\. 4. Put tube back on magnet\. 5. Wait until clear\. 6. Remove and discard LFB\. ## Wash 2 1. Add another 250 µL LFB\. 2. Remove tube from magnet\. 3. Gently resuspend beads\. 4. Put tube back on magnet\. 5. Wait until clear\. 6. Remove and discard LFB\. 7. Remove residual LFB with P10/P20\. 8. Do not overdry\. ## Elute 1. Add 25 µL EB\. 2. Remove tube from magnet\. 3. Resuspend gently\. 4. Incubate 10 minutes at room temperature\. 5. Put tube back on magnet\. 6. Wait until eluate is clear\. 7. Transfer clear eluate to a clean DNA LoBind tube labeled`Final library`\. This is the final sequencing library\. Practical rule: ``` Liquid moves. Beads stay. ``` Do not transfer beads into the final library\. ## 14\. Check quantity of DNA one more time Goal: measure final adapted library concentration\. Because low\-input runs may produce very little final library, this may read low or fail\. Use: ``` 199 µL 1X dsDNA HS working solution 1 µL final library ``` On Qubit: Record: ``` Final library concentration: Final library volume: Estimated mass loaded: ``` Calculate: ``` mass loaded ng = final library concentration ng/µL × 12 µL ``` If the final library is too low, do not repeatedly burn more library on Qubit\. For a practice run, proceed with 12 µL library in the loading mix\. ## 15\. Flow cell check with MinKNOW Goal: verify the flow cell before loading the library\. 1. Take flow cell out of fridge\. 2. Let it sit at room temperature for 20 minutes\. 3. Keep it flat\. 4. Do not shake\. 5. Plug in MinION\. 6. Open MinKNOW\. 7. Insert flow cell\. 8. Run flow cell check\. 9. Record active pores\. Decision table: ``` >1200 pores = great 800–1200 pores = usable 500–800 pores = marginal/practice <500 pores = bad, but still possible for mechanical practice <200 pores = essentially not worth it except loading practice ``` Record: ``` Flow cell ID: Starting active pores: Flow cell age: Previously used? yes/no: Washed? yes/no: ``` ## 16\. Understand the final tubes and ports At the end, there are three tubes: ``` Tube 1: Final library - This is the DNA library made after adapter cleanup. Tube 2: Priming mix - This prepares the flow cell. Tube 3: Loading mix - This contains final library + sequencing buffer + library beads. ``` There are two flow\-cell ports: ``` Port 1: Priming port - This is under the sliding cover. - Priming mix goes here. Port 2: SpotON sample port - This is the small sample well. - Loading mix goes here drop by drop. ``` Important: ``` Final library does not go directly onto the flow cell by itself. Final library first goes into the loading mix tube. ``` ## 17\. Make priming mix for ONT Flow Cell If BSA is available: ``` 1170 µL FCF 5 µL BSA 30 µL FCT Total = 1205 µL ``` If BSA is not available and this is a practice run: ``` 1170 µL FCF 30 µL FCT Total = 1200 µL ``` Do not substitute SFB, DCS, LIS, or LNB for BSA\. Mix gently\. Avoid bubbles\. This is Tube 2:`Priming mix`\. ## 18\. First prime Do this only after flow cell check is complete\. Make sure to watch this video:[https://www\.youtube\.com/watch?v=IknVaEnuDz0](https://www.youtube.com/watch?v=IknVaEnuDz0)\. It is how you load the actual flow cell\! 1. Keep flow cell plugged in and flat\. 2. Open the sliding cover to expose the priming port\. 3. Check for an air bubble near the port\. Draw back only 20–30 µL: ``` Set P1000 to 200 µL. Put tip into priming port. Slowly dial to 220–230 µL. Stop as soon as liquid enters the tip. Do not pull more. ``` Then load into the priming port: Go slowly\. Avoid bubbles\. Wait: ## 19\. Make loading mix during the 5\-minute wait Tube 3:`Loading mix` Use: ``` 37.5 µL SB 25.5 µL LIB 12 µL final library Total = 75 µL ``` Important: ``` LIB = Library Beads from the ONT kit. LIB is not AMPure XP beads. LIB settles fast. Mix LIB immediately before pipetting. ``` Steps: 1. Mix LIB immediately before pipetting\. 2. Add 37\.5 µL SB to a clean tube\. 3. Add 25\.5 µL LIB\. 4. Add 12 µL final library\. 5. Mix gently by pipetting\. 6. Do not vortex\. ## 20\. Second prime After the 5\-minute wait, load into the priming port: Avoid bubbles\. ## 21\. Load library onto SpotON port 1. Gently mix Tube 3 loading mix immediately before loading\. 2. Open the SpotON sample port\. 3. Load the full 75 µL loading mix into the SpotON port\. 4. Add it drop by drop\. 5. Let each drop disappear before adding the next\. 6. Do not force it\. 7. Do not jab the port\. 8. Avoid bubbles\. Then: ``` Close SpotON cover. Close priming port. Add light shield. Close MinION lid. Start run in MinKNOW. ``` Final mental model: ``` Priming mix → sliding priming port Loading mix → SpotON sample port Final library → only goes into loading mix first ``` ## 22\. Sequence DNA with MinKNOW Recommended basic settings: ``` Flow cell type: FLO-MIN114 Kit: SQK-LSK114 Basecalling: ON Model: High-accuracy / HAC Barcoding: OFF Alignment: OFF Adaptive sampling: OFF Raw reads / POD5: ON Filtering: OFF for low-input practice runs ``` Then: For a low\-input practice run, keep raw POD5 on so the data can be analyzed later even if live output is poor\. ## 23\. Basecall after run if needed 1. Find POD5 output directory\. 2. Install Dorado\. 3. Run basecalling: ``` dorado basecaller sup pod5_directory/ > calls.bam ``` 1. Convert BAM to FASTQ if needed: ``` samtools fastq calls.bam > reads.fastq ``` 1. For faster first pass, use HAC instead of SUP: ``` dorado basecaller hac pod5_directory/ > calls.bam ``` ## 24\. Align reads to human reference 1. Download GRCh38 FASTA\. 2. Index reference: ``` minimap2-d GRCh38.mmi GRCh38.fa ``` 1. Align reads: ``` minimap2-ax map-ont GRCh38.mmi reads.fastq | samtoolssort-o aligned.bam ``` 1. Index BAM: ``` samtools index aligned.bam ``` 1. Check alignment summary: ``` samtools flagstat aligned.bam > flagstat.txt ``` 1. Check coverage: ``` mosdepth sample_cov aligned.bam ``` ## 25\. Variant calling 1. Install Clair3\. 2. Use ONT model\. 3. Run Clair3 with GRCh38 reference, sorted BAM, and output directory\. 4. Output should include VCF\. 5. Do not overinterpret low\-coverage variants\. 6. For a first MinION run, treat this as**technical validation**, not medical\-grade interpretation\. ## 26\. Annotation 1. Install VEP\. 2. Annotate VCF against GRCh38\. 3. Add ClinVar\. 4. Add gnomAD\. 5. Add PharmGKB later\. 6. Store final table with columns: ## References [Seth Howes protocol](https://iwantosequencemygenomeathome.com/#costs)\(X profile\) [DNA Quantification with Qubit](https://www.youtube.com/watch?v=YXSaJQsIpFI) *[Molecular Biology of the Cell](https://www.amazon.com/Molecular-Biology-Sixth-Bruce-Alberts/dp/0815345240)* *[Quantifying Life](https://www.amazon.com/Quantifying-Life-Symbiosis-Computation-Mathematics/dp/022637176X)*\(super underrated\) *[Integrated Drug Discovery Technologies](https://www.amazon.com/Integrated-Drug-Discovery-Technologies-Houng-Yau-ebook/dp/B00SC81HWC/ref=sr_1_1?crid=1WQOFZZS77LE6&dib=eyJ2IjoiMSJ9.-1b9qnZYOTdD8F9n4EqjyU2xLAWmJwd14zom3XbYxoKZcdJ8J0qpIsZD_jz-DtzgbjsZufP2mK-DYvWTYH8OYzAqsoGHgCkkJ697picfAtb_tCD2aYhtMHof_cIcnZvI8p_cvEyzmsKfmvoc1nB2G925qblja1OGOO6n_DNn-A7ACBHw8ci2zY-iB4u0fTNhZlR5jQHFDz0grGrLzpEYMTpOAJKPNAGv6PWpPBJK6JY.ngjMGi_K9Q-HCSpN4u8nIWhq5LncwL2MB4JBvJYlObY&dib_tag=se&keywords=integrated+drug+discovery+technologies&qid=1781302527&sprefix=integrated+drug+d%2Caps%2C230&sr=8-1)* *[Darkome](https://www.amazon.com/Darkome-Hannu-Rajaniemi-ebook/dp/B0C9GNJH1C)* *[Upgrade](https://www.amazon.com/Upgrade-Novel-Blake-Crouch/dp/0593157532)* [Sid’s cancer data](https://osteosarc.com/) [DNA Whole Genome Sequencing \- astrology for sigmas](https://vad1ms.substack.com/p/dna-whole-genome-sequencing-astrology?r=2xxyvi&utm_campaign=post&utm_medium=web&triedRedirect=true) [DNA sequencing at home with the flongle](https://blog.booleanbiotech.com/sequencing-at-home-with-flongle)