Aug 05, 2025

Public workspaceGETCampy Sequencing protocols

DOI
https://dx.doi.org/10.17504/protocols.io.e6nvw4jpwlmk/v1
GETCampy Sequencing protocols
  • Ben Pascoe1,
  • Henry Badji2,
  • Madison Goforth1,
  • Evangelos Mourkas3,
  • Evariste Bako4,
  • Isidore Bonkoungou4,
  • Modeste T Gampene4,
  • M Edith M Nikiema4,
  • Barthélemy Zoma4,
  • Ousman E Bah2,
  • Eustacia J Cassell2,
  • Abdoulie K Ceesay2,
  • Bubcarr E Ceesay2,
  • Ousman Ceesay2,
  • Ousman Ceesay2,
  • Bakary Conteh2,
  • Lamin Drammeh2,
  • Binta Faye2,
  • Abdoulie F Jalllow2,
  • Fatima Jallow2,
  • Ousman Jallow2,
  • Samba Juma Jallow2,
  • Pa Modou Joof2,
  • Modou Kandeh2,
  • Mehrab Karim2,
  • Jarra Manneh2,
  • Abdou Ceesay5,
  • Ebrima Fofana5,
  • Mamud Jallow5,
  • Modou B Jarju5,
  • Samba Ba6,
  • Dodou Sanyang6,
  • Demba B Jallow7,
  • Mukaila I Alebiosu8,
  • Akosua Bonsu Karikari8,
  • Atanyiwoen Brusah9,
  • Courage KS Saba9,
  • Kaisa Haukka10,
  • Catherine Matilda Collins11,
  • Adrian W Leach11,
  • Polina Levontin11,
  • Shani UP Ali12,
  • Frances M Colles13,
  • Matthew Hitchings13,
  • Martin Antonio1,
  • Abdul Karim Sesay1,
  • Ozan Gundogdu14,
  • Jahangir Hossain2,
  • Samuel K Sheppard1,
  • Bubacarr E Ceesay15,
  • Bubacarr E. Ceesay16
  • 1Ineos Oxford Institute for Antimicrobial Research, Department of Biology, University of Oxford, Oxford, United Kingdom;
  • 2Medical Research Council (UK) Unit The Gambia at the London School of Hygiene and Tropical Medicine, Banjul, The Gambia;
  • 3Zoonosis Science Center, Department of Medical Sciences, Uppsala University, Uppsala, Sweden;
  • 4Laboratory of Molecular Biology, Epidemiology and Monitoring of Bacteria and Virus Transmitted by Food (LaBESTA), University Joseph KI-ZERBO, 03 BP 7021 Ouagadougou 03, Burkina Faso;
  • 5Department of Livestock Services, Banjul, The Gambia;
  • 6Regional health Directorate, Upper River Region, Ministry of Health, The Gambia;
  • 7National Agricultural Research Institute, The Gambia;
  • 8Department of Clinical Microbiology, School of Medicine, University for Development Studies, Tamale, Ghana;
  • 9Faculty of Biosciences, Department of Biotechnology. University for Development Studies, Tamale,Ghana;
  • 10Department of Microbiology, University of Helsinki, Helsinki, Finland;
  • 11Centre for Environmental Policy, Imperial College London, The Weeks Building, 16-18 Princes Gardens, London SW7 1NE, United Kingdom;
  • 12Milner Centre for Evolution, Department of Life Sciences, University of Bath, Bath, UK;
  • 13Department of Biology, University of Oxford, Oxford, United Kingdom;
  • 14Department of Infection Biology, Faculty of Infectious & Tropical Diseases, London School of Hygiene and Tropical Medicine;
  • 15Medical Research Council at the London School of Hygiene and Tropical Medicine;
  • 16Medical Research Council Unit, The Gambia, at London School of Hygiene and Tropical Medicine
  • GETcampy
Ben Pascoe
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DOI: https://dx.doi.org/10.17504/protocols.io.e6nvw4jpwlmk/v1
External link: https://sheppardlab.wordpress.com/projects/get-campy-africa/
Protocol Citation: Ben Pascoe, Henry Badji, Madison Goforth, Evangelos Mourkas, Evariste Bako, Isidore Bonkoungou, Modeste T Gampene, M Edith M Nikiema, Barthélemy Zoma, Ousman E Bah, Eustacia J Cassell, Abdoulie K Ceesay, Bubcarr E Ceesay, Ousman Ceesay, Ousman Ceesay, Bakary Conteh, Lamin Drammeh, Binta Faye, Abdoulie F Jalllow, Fatima Jallow, Ousman Jallow, Samba Juma Jallow, Pa Modou Joof, Modou Kandeh, Mehrab Karim, Jarra Manneh, Abdou Ceesay, Ebrima Fofana, Mamud Jallow, Modou B Jarju, Samba Ba, Dodou Sanyang, Demba B Jallow, Mukaila I Alebiosu, Akosua Bonsu Karikari, Atanyiwoen Brusah, Courage KS Saba, Kaisa Haukka, Catherine Matilda Collins, Adrian W Leach, Polina Levontin, Shani UP Ali, Frances M Colles, Matthew Hitchings, Martin Antonio, Abdul Karim Sesay, Ozan Gundogdu, Jahangir Hossain, Samuel K Sheppard, Bubacarr E Ceesay, Bubacarr E. Ceesay 2025. GETCampy Sequencing protocols. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvw4jpwlmk/v1
Manuscript citation:
TBC
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: In development
We are still developing and optimizing this protocol
Created: August 05, 2025
Last Modified: August 05, 2025
Protocol Integer ID: 224086
Keywords: Campylobacter, gastroenteritis, genomics, pathogen surveillance, Whole genome sequencing, 16S sequencing, metagenomics sequencing, genomic surveillance of campylobacter, other enteric pathogens across africa, getcampy sequencing protocol, getcampy sequencing protocols as part, enabling hybrid metagenomic approach, analysing enteric pathogen, assembled genome, hybrid metagenomic approach, enteric pathogen, resolution genome assembly, other enteric pathogen, genomic surveillance, sequencing method, campylobacter, dna extraction protocol, taxonomic profiling, total community dna, oxford nanopore, high molecular weight recovery,
Funders Acknowledgements:
Medical Research Council (UK)
Grant ID: MR/V001213/2
Disclaimer
Protocols for research purposes only
Abstract
As part of the GETCampy-Africa project, this protocol provides a modular workflow for extracting, sequencing, and analysing enteric pathogens from stool and environmental samples. It includes short-read (Illumina MiSeq) and long-read (Oxford Nanopore) sequencing methods, enabling hybrid metagenomic approaches for high-resolution genome assembly. DNA extraction protocols support both total community DNA and high molecular weight recovery. Library preparation workflows include Nextera XT (Illumina), rapid barcoding and ligation kits (ONT), and full-length 16S rRNA sequencing for taxonomic profiling. The protocol also outlines hybrid assembly, binning, and quality control steps for metagenome-assembled genomes (MAGs). Designed for field-lab compatibility and resource-limited settings, these methods support genomic surveillance of Campylobacter and other enteric pathogens across Africa.
Image Attribution
CCBY c/o Ben Pascoe
Troubleshooting
DNA extraction
🧬 QIAamp DNA Extraction Protocol for Campylobacter
Kit: QIAGEN QIAamp DNA Mini Kit Adapted from: QIAamp DNA Mini Handbook, Appendix D (page 55) and Step 3 (page 33) Organism: Campylobacter spp.
🧫 Sample Preparation
  1. Grow Campylobacter isolates:
  • Medium: Chocolate, blood, or Brucella agar
  • Conditions: 42°C for 48 hours
Using a sterile loop, harvest at least one loop-full of bacterial growth.
Resuspend cells in 180 µL Buffer ATL.
Incubate at 37°C for 30 minutes.
🧪 Cell Lysis and Protein Digestion
Add 20 µL Proteinase K (from the kit).
Mix thoroughly by vortexing.
Incubate at 56°C for 30–60 minutes.
Briefly spin down to remove condensation from the tube lid.
🔬 RNase Treatment and Lysis Enhancement
Add:
  • 4 µL RNase (100 mg/mL)
  • 200 µL Buffer AL (from the kit)
Vortex to mix completely.
Incubate at 70°C for 10 minutes.
💧 DNA Binding
Add 200 µL of 95–100% ethanol to the lysate.
Vortex to mix.
Transfer the full volume (~600 µL) into a QIAamp spin column in a 2 mL collection tube.
Centrifuge at 8000 rpm for 1 minute at room temperature (RT).
Discard the flow-through and move the column to a clean 2 mL collection tube.
🚿 Wash Steps
Add 500 µL Buffer AW1 to the column.
Centrifuge at 8000 rpm for 1 minute at RT.
Discard flow-through.
Add 500 µL Buffer AW2.
Centrifuge at 14,000 rpm for 3 minutes to dry the membrane.
💦 DNA Elution
Place the QIAamp column into a clean 1.5 mL microcentrifuge tube.
Add 50 µL Buffer AE (or nuclease-free water) directly to the column membrane.
Let sit at room temperature for 1 minute.
Centrifuge at 8000 rpm for 1 minute to elute DNA.
📦 Storage
  • Short-term: Store at 4°C
  • Long-term: Store at -20°C
📝 Notes
  • Ensure all reagents from the kit are at working temperature.
  • Eluted DNA is suitable for downstream applications including PCR and sequencing.
Library preparation for short read sequencingUntitled section
🧬 Illumina Custom Protocol (Nextera XT on MiSeq)
Instrument: MiSeq Library Prep: Nextera XT DNA Reagent Kit: MiSeq v3 Indexing: Dual Indexing Software: BaseSpace Sequence Hub (FASTQ generation)
📋 Generate Sample Sheet with IEM
Start IEM Software
  • Start → All Programs → Illumina → Illumina Experiment Manager or click IEM icon.

Create Sample Plate
Select Create Sample Plate → Choose Nextera XT.
Set:
  • Unique plate name
  • Index reads = 2

Enter sample details per well (ID, i7/i5 index, etc.)
Save as *.nexxt28.plt
Create Sample Sheet
Select Create Sample Sheet → Instrument: MiSeq.
Application: FASTQ Only → Click Next.
Set:
  • Reagent barcode
  • Prep kit: Nextera XT
  • Index reads: 2
  • Experiment name, date, paired/single-end, cycles
Load plate, select samples, and save sample sheet.
🧪 Nextera XT Library Prep
Tagment Genomic DNA
  • Thermal cycler: 55°C for 5 min, hold at 10°C
  • Mix in PCR plate:
  • 10 µL TD + 5 µL gDNA (0.2 ng/µL)
  • Add 5 µL ATM, mix
  • Spin 280 × g, 1 min → Run tagmentation
  • Add 5 µL NT, mix → Spin → RT 5 min
Amplify Libraries
  • Thermal cycler:
  • 72°C 3 min → 95°C 30s
  • 12 cycles: 95°C 10s → 55°C 30s → 72°C 30s
  • Final: 72°C 5 min → hold at 10°C
  • Add i7 and i5 indices (5 µL each), then 15 µL NPM
  • Spin → Run PCR
Safe stop: 2–8°C for 2 days or overnight on thermal cycler
🧼 Clean Up Libraries
Transfer 50 µL PCR product to new plate.
Add 30 µL AMPure XP beads → Shake 1800 rpm 2 min → RT 5 min
Magnetic stand → remove supernatant.
Wash ×2 with 200 µL 80% EtOH → Remove residual EtOH
Air dry 15 min.
Elute in 52.5 µL RSB → Shake, incubate, magnet, transfer 50 µL to new plate
Safe stop: -25°C to -15°C for 7 days
🔍 Check Libraries (Optional)
  • Run 1 µL on Agilent Bioanalyzer (HS DNA chip)
⚖️ Normalize Libraries
Transfer 20 µL to new plate.
Mix 4.4 mL LNA1 + 800 µL LNB1 → Add 45 µL per well
Shake 1800 rpm 30 min → Magnet → Remove supernatant
Wash ×2 with 45 µL LNW1
Add 30 µL 0.1 N NaOH → Shake 5 min
Add 30 µL LNS1 to new plate (SGP)
Resuspend beads → Shake 5 min → Magnet → Transfer to SGP plate
Spin at 1000 × g for 1 min
Safe stop: -25°C to -15°C for 7 days
🧬 Pool Libraries
Spin SGP plate.
Transfer 5 µL per sample to PCR tube strip → Pool into tube (PAL)
Dilute to loading concentration.
Store PAL and SGP: -25°C to -15°C (7 days)
🧊 Denature and Dilute Libraries
Prepare HT1
  • Thaw at RT, store at 2–8°C.
Dilute Library

Library VolumeHT1 Volume
6 µL594 µL
24 µL (XT)576 µL

  • Vortex → Spin → Incubate 98°C 2 min → Ice 5 min
Illumina short read sequencing
‱ C-SOP-601: Illumina MiSeq Operation for Whole Genome Sequencing (WGS)
‱ Preparation Before Starting
Clean workspace with 80% ethanol.
Wear PPE and follow local GLP guidelines.
Prepare ice bucket for temporary reagent/sample storage.
Thaw HT1 buffer at room temperature, then store at 4°C until use.
Prepare 0.2N NaOH:
- Mix 200 µL 1.0N NaOH + 800 µL nuclease-free water.
- Use within 8 hours.

‱ Reagent Cartridge Thawing (2 Hours Prior to Run)
Remove cartridge from -20°C.
Submerge base only in room-temp water bath (max line indicated).
Thaw time:
- v3 kits: ~1.5 hours
- v2 kits: ~1 hour
Dry base and invert 10x to mix reagents.
Check positions 1, 2, and 4 for full thawing (no precipitates).
Store at 4°C until ready to load.

‱ MiSeq Instrument Initialisation
Power on MiSeq (switch on back).
Log in to BaseSpace, if networked.
Wait for MiSeq Control Software (MCS) to initialise.

‱ Denature and Dilute DNA Library
Mix:
- 5 µL 4nM DNA library
- 5 µL 0.2N NaOH
Vortex 10 sec, spin 1 min.
Incubate 5 min at RT.
Add 990 µL chilled HT1 → final 20pM denatured library.
Dilute to final loading concentration (e.g., 10pM).

‱ Denature and Dilute PhiX (Optional Spike-in)
Dilute 2 µL 10nM PhiX + 3 µL Tris-Cl/Tween → 4nM.
Mix:
- 5 µL 4nM PhiX
- 5 µL 0.2N NaOH
Vortex 10 sec, spin 1 min, incubate 5 min.
Add 990 µL HT1 → final 20pM PhiX.
For v2 kits: mix 375 µL PhiX + 225 µL HT1 → 12.5pM.
Add 1–5% PhiX to final sample mix depending on library complexity.

‱ MiSeq Run Setup
Ensure ≥100 GB free disk space.
Enable local analysis replication:
- MCS → Run Options → check "Replicate analysis locally".
Prepare and load Sample Sheet via Illumina Experiment Manager (IEM).

‱ Prepare and Load Flow Cell
Retrieve Flow Cell + PR2 buffer from 4°C.
Rinse flow cell with lab-grade water → dry with lint-free lens paper.
Clean glass with ethanol-wetted lens tissue.
Install flow cell into clamp → wait for RFID recognition.

‱ Load Incorporation Buffer (PR2) and Empty Waste
Raise sipper handle.
Load mixed PR2 buffer bottle → confirm RFID.
Empty and replace waste bottle.
Lower sipper into PR2 + waste bottle.

‱ Load DNA Library into Cartridge
Tap cartridge to gather contents.
Pierce foil at "Load Sample".
Pipette 600 µL denatured library into well.
Tap gently to collect sample at base.

‱ Load Cartridge into MiSeq
Wait for “Load Reagent Cartridge” prompt.
Remove wash tray, dry chiller base if needed.
Insert cartridge (Illumina label facing out) until fully seated.
Confirm cartridge RFID is read.

‱ Sample Sheet Confirmation
MCS looks for matching barcode sample sheet.
If not found, manually browse and select.
Click “Save and Continue” → “Next”.

‱ Start Sequencing Run
Review:
- Experiment Name
- Workflow
- Read Length
Perform Pre-run Check.
When all checks pass, click “Start Run”.

‱ Post-Run Wash (Required)
Follow on-screen prompts to load:
- Wash tray
- Wash bottle
- Optional: MiSeq wash tube (0.01% NaOCl for bleach wash)
Start Wash.
Do not remove flow cell/wash tray during wash.
Leave wash solution in tray/bottle after wash completes.

‱ Monitor Run
- Use Sequencing screen (view-only) or BaseSpace/SAV.
- Monitor: cycles, cluster intensities, quality scores.

‱ Export Data
- Navigate to:
`D:\Illumina\MiSeq Output\Run Folder\Data\Intensities\BaseCalls`
- Download `fastq.gz` files from R1 and R2.
- Or use BaseSpace/local network share.

‱ Instrument Shutdown or Reboot
- Weekly reboot recommended
- MCS → Manage Instrument → Reboot
- For shutdown:
- MCS → Manage Instrument → Shutdown
- Power switch off → wait 10 min → power on

‱ Monthly Maintenance
Week | Action
1 PTL (post-run bleach)
2 PTL
3 PTL
4 PTL or Maintenance
- See MiSeq System Guide for detailed cleaning steps.

‱ Troubleshooting Resources
- MiSeq System Guide
- Sample Sheet Quick Guide
- MiSeq Reporter / SAV Software Guide
- Re-Queue Tutorials
High molecular weight DNA extraction
🧬 C-SOP-1001: HMW DNA Extraction from Bacteria Using NEB Monarch HMW DNA Extraction Kit

🔧 Materials Required (Not Supplied)
  • Microcentrifuge
  • Thermal mixer (1.5 mL + 2 mL blocks)
  • Vertical rotating mixer (e.g., HulaMixer)
  • Ethanol ≥95%
  • Cold PBS or TE/Tris
  • Isopropanol (550 µL/sample or 275 µL low input)
  • 1.5 mL DNase-free low DNA binding tubes
  • Wide-bore pipette tips
  • Lysozyme (Gram-neg: 25 mg/mL; Gram-pos: 10 mg/mL in STET)
  • Additional lysis agents (e.g., lysostaphin if needed)

🧤 Preparation
Clean bench with 80% ethanol. Wear PPE.
Preheat thermal mixers to 37°C and 56°C.
Add ethanol to gDNA Wash Buffer as per label.
Chill PBS/TE/Tris buffer.
Label tubes. Store Proteinase K and RNase A at -20°C.

🧫 Step 1: Cell Pellet
  • Centrifuge bacterial culture at 12,000 × g for 1 min in a Monarch Pestle Tube.

🦠 Step 2: Lysis
A. Gram-negative Bacteria
Resuspend pellet in 300 µL cold PBS (150 µL for low input).
Add 10 µL lysozyme (25 mg/mL), vortex briefly.
Add 300 µL HMW Lysis Buffer, invert 5–10×.
Incubate at 37°C with agitation:
  • 2000 rpm (faster lysis, shorter fragments)
  • 500 rpm (slower lysis, longer fragments)
  • Until lysate turns clear (~5 min)

B. Gram-positive Bacteria
Resuspend in STET buffer + lysozyme (10 mg/mL).
Incubate 30 min at 37°C (no agitation).
Add 300 µL HMW Lysis Buffer, invert 5–10×.

🔥 Step 3: Proteinase K Digestion
Increase thermal mixer to 56°C if needed.
Add 20 µL Proteinase K (10 µL for low input), invert 10–20×.

Choose Homogenization:
  • Thermal Mixer: Incubate 30 min at 56°C, 2000 rpm.
  • Rotor-stator: Homogenize 5–15 sec, then incubate 30 min at 56°C.

💧 Step 4: RNA Removal
Add 10 µL RNase A (5 µL for low input), invert 5–10×.
Incubate 10 min at 56°C, same speed as lysis.

🧪 Step 5: Protein Separation
Add 300 µL Protein Separation Solution, invert for 1 min or mix at 20 rpm.
Centrifuge 10 min at 16,000 × g.
Carefully transfer upper DNA phase (~800 µL) to new 2 mL tube.

🧬 Step 6: DNA Binding to Beads
Add 2 DNA Capture Beads to tube.
Add 550 µL isopropanol (275 µL low input).
Mix gently:
  • Rotate at 10 rpm for 5 min OR
  • Invert 25–30× slowly (5–6 sec per inversion)
  • Watch for visible DNA wrapping around beads

🚿 Step 7: Wash Steps
Remove liquid carefully (avoid touching beads).
Wash with 500 µL gDNA Wash Buffer, invert 2–3×, then remove.
Repeat wash.

🧲 Step 8: Bead Transfer
Transfer beads to Monarch Collection Tube II + bead retainer.
Pulse spin (≤1 sec).
Move beads to a fresh 2 mL tube.
Insert bead retainer into 1.5 mL DNA low-bind tube.

💦 Step 9: Elution
Add 100 µL Elution Buffer II (≥5 min at 56°C, 300 rpm).
  • Optional: use 50 µL with more frequent gentle shaking
Pour eluate + beads into bead retainer in 1.5 mL tube.

🌀 Step 10: Final Handling
Centrifuge 30 sec at 12,000 × g.
Discard beads and retainer.
Pipette eluate 5–10× with wide-bore tips to disperse aggregates.
Dissolve DNA:
  • 1 h at 37°C, or
  • 24 h at RT, or
  • Overnight at 4°C

Pipette again before use.

📦 Storage
  • Short-term: 4°C
  • Long-term: -20°C (in Elution Buffer: 10 mM Tris, pH 9.0, 0.5 mM EDTA)

📚 Additional Resources
Rapid barcoding for long read sequencing
🧬 Rapid Barcoding Workflow
Kit: ONT Rapid Barcoding Kit 96 (SQK-RBK114.96) Platform: MinION / GridION / PromethION Purpose: For barcoded, multiplexed sequencing of DNA using ONT’s transposase-based tagmentation
🧪 DNA Input Requirements
  • Input amount: 10–50 ng per sample (in ≤2.5 µL nuclease-free water)
  • Volume of RB reagent per sample: 2.5 µL
  • Volume of barcode (BCXX) per sample: 0.5 µL
  • Multiplexing: Up to 96 samples
🔬 Barcoding Reaction
Prepare Reaction Mix
In a PCR strip tube or plate (per sample):

ReagentVolume (µL)
Genomic DNA≤2.5
RB (Fragmentation Mix)2.5
Barcode (BCXX)0.5
Total5.5 max

Mix and Incubate
  • Gently flick or pipette to mix.
  • Incubate at 30°C for 1 minute, then 80°C for 1 minute.
  • Immediately place on ice.
🧹 Pooling and Cleanup
Pool 2 µL from each barcoded sample into a single tube.
Total volume should not exceed 10 µL.
Proceed to magnetic bead cleanup:
  • Add 1.8× volume of AMPure XP beads.
  • Incubate 5 minutes at RT.
  • Place on magnet for 5 minutes.
  • Wash twice with 70% ethanol.
  • Elute in 10 µL Elution Buffer or nuclease-free water.
🧪 Adapter Ligation

ReagentVolume (µL)
Barcoded DNA10.0
RAP (Rapid Adapter)1.0
Total11.0

  • Mix by flicking or pipetting.
  • Incubate at room temperature for 5 minutes.
  • Proceed immediately to loading.
💉 Loading onto Flow Cell
  • Load the library onto a primed flow cell following the Flow Cell Priming protocol.
  • Use appropriate loading buffer and loading volume as per the platform used (MinION/GridION/PromethION).
  • Avoid bubbles when loading.
🧊 Storage and Stability
  • Barcoded samples: Use immediately or store at -20°C (short-term).
  • Final library: Load immediately. Do not freeze post-adapter ligation.
Long read sequencing using ONT
🧬 Long Read Sequencing Protocol using Oxford Nanopore Technologies
Workflow: Ligation Sequencing Kit (LSK114) Platform: MinION / GridION / PromethION Purpose: For high-yield, long-read sequencing using ONT flow cells
🧪 Input DNA Requirements
  • Amount: ≥1 µg high molecular weight DNA
  • Concentration: ≥50 ng/µL in ≥20 µL volume
  • Buffer: Nuclease-free water or low-salt TE (avoid EDTA >0.1 mM)
  • Fragmentation: Avoid shearing to preserve read length
🔬 DNA Repair & End-Prep

ReagentVolume (µL)
DNA sampleUp to 48.0
NEBNext FFPE DNA Repair Buffer7.0
NEBNext FFPE DNA Repair Mix3.0
NEBNext Ultra II End Prep Reaction Buffer3.5
NEBNext Ultra II End Prep Enzyme Mix3.0
Total~64.5

Mix gently, spin briefly.
Incubate:
  • 20°C for 5 min
  • 65°C for 5 min
Place on ice.
🧲 AMPure XP Bead Cleanup
Add 1× volume (e.g., 65 µL) of AMPure XP beads.
Incubate 5 min at RT.
Magnet for 5 min → remove supernatant.
Wash twice with 200 µL 70% ethanol.
Air dry beads for 2 min (avoid over-drying).
Elute DNA in 61 µL nuclease-free water.
🔗 Adapter Ligation

ReagentVolume (µL)
Eluted DNA60.0
NEBNext Quick T4 DNA Ligase Buffer (5X)25.0
Adapter Mix (AMX)10.0
Quick T4 DNA Ligase5.0
Total100.0

Mix by gentle flicking.
Incubate room temperature for 10–15 minutes.
🧲 Post-Ligation Cleanup
Add 0.4× volume (~40 µL) of AMPure XP beads.
Incubate 5 min at RT.
Magnet for 5 min → discard supernatant.
Wash twice with 250 µL Long Fragment Buffer (LFB).
Air dry beads (≤2 min).
Elute final library in 15 µL Elution Buffer (EB).
💧 Flow Cell Priming (MinION/GridION)
Thaw and mix:
  • Flush Buffer (FB)
  • Flush Tether (FLT) → mix with FB to make priming mix
Open SpotON port and priming port.
Remove ~20 µL from priming port to release air.
Slowly inject 800 µL priming mix via priming port.
💉 Load Library
Prepare loading mix:
  • 37.5 µL Sequencing Buffer (SQB)
  • 25.5 µL Loading Beads (LB, mix just before use)
  • 12 µL Final library (from step 5)
Mix gently by pipetting.
Load 75 µL into the SpotON port drop-by-drop.
Close ports and lid.
▶️ Start Sequencing Run
Use MinKNOW software.
Choose appropriate flow cell and kit settings (e.g., LSK114).
Set run time (e.g., 24–72 hours depending on goal).
Start sequencing and monitor real-time QC metrics.
🧼 After the Run
  • Optionally wash and reuse flow cells using Flow Cell Wash Kit (up to 2–3 times).
  • Store remaining library at -20°C for short-term re-use (avoid freeze-thaw).
✅ Optional Tips
  • For ultra-long reads, minimize pipetting and vortexing.
  • Use wide-bore or cut tips during extraction and library prep.
  • Consider using Qubit and Femto Pulse for DNA QC and sizing.
16S sequencing for sample taxonomy profiling
🧬 16S rRNA Gene Amplicon Sequencing (ONT Workflow)
Goal: Taxonomic profiling via full-length 16S rRNA gene sequencing Platform: MinION / GridION / PromethION Kit: 16S Barcoding Kit (SQK-16S024 or similar) Read Type: Long-read, full-length 16S (~1.5 kb)
🧫 Sample Preparation
  • Extract total DNA from microbial or environmental samples.
  • Use a high-fidelity, contamination-free method (e.g., Qiagen PowerSoil for stool).
  • Quantify DNA using Qubit (dsDNA HS).
  • Normalize DNA to 10–50 ng per PCR reaction.
🔁 16S rRNA Gene PCR Amplification

ReagentVolume (µL)
Template DNA (10–50 ng)~2–5
Barcoded 16S primers (forward + reverse)1.0
LongAmp Taq 2X Master Mix (NEB)25.0
Nuclease-free waterto 50.0

Cycling Conditions (for full-length 16S):
  • 95°C for 3 min
  • 25 cycles of:
  • 95°C for 20 sec
  • 55°C for 30 sec
  • 65°C for 2 min
  • Final: 65°C for 5 min → hold at 4°C
✅ Tip: Use primers covering full-length 16S gene (e.g., 27F/1492R).
🧲 Clean Up PCR Products
  • Use AMPure XP beads (1× ratio):
  • Mix, incubate 5 min RT
  • Magnet, wash ×2 with 70% EtOH
  • Elute in 10 µL nuclease-free water
🧬 Pooling and Quantification
  • Pool 10 µL of each barcoded sample.
  • Quantify final pool via Qubit.
  • Aim for 50–200 fmol total DNA (up to 1 µg).
🔗 Adapter Ligation (if not pre-tagged)

ReagentVolume (µL)
Pooled amplicons~10
Rapid Adapter (RAP) or AMX1.0
Incubate5 min at RT

💉 Load Library onto Flow Cell
Prime the flow cell (see ONT Flow Cell Priming SOP).
Prepare the loading mix:
  • ~12 µL DNA library
  • 25.5 µL Loading Beads (LB)
  • 37.5 µL Sequencing Buffer (SQB)
Mix gently and load 75 µL dropwise onto SpotON port.
▶️ Start Sequencing
  • Use MinKNOW and select:
  • Kit: SQK-16S024 (or latest)
  • Flow Cell: FLO-MIN106D (R10.4.1 recommended)
  • Duration: 12–48 hours depending on depth
🧠 Data Analysis
  • Basecalling via Guppy or MinKNOW
  • Taxonomic classification:
  • Use EPI2ME Fastq 16S Workflow, or
  • Tools like QIIME 2, Kraken2, or NanoCLUST
  • Generate bar plot of relative abundances per sample
📦 Storage
  • Store final amplicon libraries at -20°C short-term.
  • Flow cells can be washed and reused (1–2 times).
✅ Notes
  • Full-length 16S sequencing provides species-level resolution.
  • Minimize chimera formation by using high-fidelity polymerases.
  • Always run negative controls to detect contamination.
Hybrid sequencing for metagenomes
🧬 Hybrid Metagenomic Sequencing Protocol (Illumina + ONT)
📦 Sample Collection & DNA Extraction
Collect samples (e.g. stool, soil, rumen, etc.) under sterile conditions.
Store samples at −80°C if not processing within 1 hour.
Extract high molecular weight (HMW) DNA using a suitable kit (e.g. QIAGEN MagAttract HMW DNA Kit).
  • Minimise mechanical shearing.
  • Aim for fragment sizes >20 kb for ONT compatibility.
Quantify DNA with Qubit and assess quality on TapeStation or agarose gel.
🧪 Library Preparation
Illumina (Short-Read) Library Prep
  • Input: ~100 ng DNA.
Recommended kits:
  • Illumina Nextera XT, or
  • NEBNext Ultra II FS.
Fragmentation: ~300–500 bp.
Add adapters and indices.
Clean up with AMPure XP beads.
Validate size distribution with Bioanalyzer or similar.
ONT (Long-Read) Library Prep
  • Input: ~1–2 µg of high-quality HMW DNA.
Recommended kits:
  • Ligation Sequencing Kit (SQK-LSK114) – highest accuracy.
  • OR Rapid Sequencing Kit (SQK-RBK114.96) – faster prep.
Perform end-repair/dA-tailing if needed.
Ligate adapters.
Clean up and elute DNA for sequencing.
🧬 Sequencing
Illumina
  • Platform: HiSeq, NovaSeq or NextSeq.
  • Paired-end 150 bp reads.
  • Target depth: ~20–40 Gbp per sample.

ONT
  • Platform: MinION, GridION, or PromethION.
  • Flowcell: R10.4.1 or latest.
  • Target depth: ~5–10 Gbp per sample.
  • Monitor in real-time, reload flow cell if necessary.
🧮 D. Data Processing & Hybrid Assembly
Quality Control
  • ONT: Basecall using Guppy (super accuracy), filter reads < Q10.
  • Illumina: Trim adapters and low-quality bases using fastp or TrimGalore

Assembly
Short-read only:
  • metaSPAdes
  • MEGAHIT
Long-read only:
  • Flye
  • Raven
Hybrid assembly:
  • metaFlye
  • HybridSPAdes
  • OPREA-MS

Polishing
Use
  • Racon (long reads)
  • Pilon (short-read) for iterative polishing.

🧱 Binning & Genome Reconstruction
Contig Binning Tools:
  • MetaBAT2
  • MaxBin2
  • VAMB
  • SemiBin
Inputs: k-mer composition and coverage profiles.

Bin Refinement
Merge/refine bins with
  • DAS Tool
  • MetaWRAP

MAG Quality Assessment
Use to assess completeness and contamination.
  • CheckM
  • BUSCO

Taxonomic Assignment
Annotate bins using
  • GTDB-Tk
🧬 Downstream Analysis (Optional)
Functional annotation:
  • PROKKA / BAKTA
  • eggNOG-mapper

Phylogenomics:
  • PhyloPhlAn

AMR/virulence:
  • ABRicate
  • CARD
  • VfDB
  • AMRfinder Plus
💡 Best Practices & Notes
  • Aim for >40 Gbp of Illumina data per sample for deep profiling.
  • ONT long reads help resolve repeats, rRNA operons, plasmids, and MGEs.
  • For low-complexity samples, less sequencing depth may suffice.
  • Optional: Use Hi-C or read cloud technologies for improved binning.