Jun 03, 2025
Version 3
REDI-NET B-4 BLOOD TESTING V.3
- REDI-NET Consortium1
- 1REDI-NET Consortium
- Remote Emerging Disease Intelligence - NETwork (REDI-NET)
Protocol Citation: REDI-NET Consortium 2025. REDI-NET B-4 BLOOD TESTING. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzbze8vx1/v3Version created by REDI-NET Consortium
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: March 19, 2025
Last Modified: June 03, 2025
Protocol Integer ID: 124633
Keywords: SEQUENCING LIBRARY PREPARATION, ONT Native barcoding kit v14, cDNA sequencing, TNA sequencing, Total nucleic acid, gDNA sequencing, Blood samples, Whole genome shotgun sequencing, nanopore sequencing, sequencing read, oxford nanopore, pathogen detection, reads for downstream data analysis, sequencing
Funders Acknowledgements:
USAMRAA
Grant ID: W81XWH-21-C-0001
USAMRAA
Grant ID: W81XWH-22-C-0093
USAMRAA
Grant ID: HT9425-23-C-0059.
USAMRAA
Grant ID: HT9425-24-C-0072
Disclaimer
This work is supported by the US Army Medical Research and Development Command under Contract No.W81XWH-21-C-0001, W81XWH-22-C-0093, HT9425-23-C-0059 and HT9425-24-C-0072. The views, opinions and/or findings contained in this report are those of the author(s) and should not be construed as an official Department of the Army or Navy position, policy or decision unless so designated by other documentation.
Abstract
This SOP provides guidance on procedures of Oxford Nanopore sequencing to generate sequencing reads for downstream data analysis and pathogen detection.
Guidelines
OBJECTIVE
To outline the procedures for properly using the Oxford Nanopore Sequencing platforms (GridION/MinION: MinION flow cell or P2 Solo: PromethION flow cell) to sequence DNA and cDNA extracted from collected blood samples.
SUMMARY/SCOPE
This SOP provides guidance on procedures of Oxford Nanopore sequencing to generate sequencing reads for downstream data analysis and pathogen detection.
RESPONSIBLE PERSON
Principal Investigator, Study Coordinator, Entomology Component Lead, Managers
Note
NOTE: All study procedures must be conducted in compliance with national and local policies for the prevention and control of COVID-19 infection.
MAINTENANCE OF EQUIPMENT
CAUTION ON RNA HANDLING:
- RNases are very stable and difficult to inactivate and only minute amounts are sufficient to destroy RNA.
- Care should be taken to avoid inadvertently introducing RNases into the samples during or after the purification procedure.
- Clean the work surfaces with RNA Zap to remove nucleases, then wipe the surfaces with 70% to 100% molecular biology grade ethanol to remove additional contaminants.
HANDLING ENZYMATIC REACTIONS
Reagents containing enzymes should be handled On ice before mixed and transferred to the assigned activation temperature.
REFERENCES
REDI-NET Overview Summary
cDNA synthesis and amplification:
● Claro IM, Ramundo MS, Coletti TM et al. Rapid viral metagenomics using
SMART-9N amplification and nanopore sequencing. Wellcome Open
Research 2023, 6:241. https://doi.org/10.12688/wellcomeopenres.17170.2
Oxford Nanopore Manufacturer's protocols:
- Ligation sequencing gDNA - Native Barcoding Kit 96 V14 (SQK-NBD114.96)-minion.
- ligation-sequencing-gdna-native-barcoding-v14-sqk-nbd114-96-NBE_9171_v114_revG_15Sep2022-minion
- ligation-sequencing-gdna-native-barcoding-v14-sqk-nbd114-96-NBE_9171_v114_revG_15Sep2022-gridion
Host rRNA depletion
- Manual: riboPOOL Kit
Host DNA depletion
- Instruction manual: NEBNext® Microbiome DNA Enrichment Kit
APPENDICES
APPENDIX 2. FLOW CELL
MinION flow cell:
PromethION flow cell:
APPENDIX 3. MinKNOW SOFTWARE INSTRUCTIONS
After Double-clicking the MinKNOW icon, the login page should show up. Use Oxford Nanopore Community username and password to login.
Select device shown on the screen.
Go to Start tab, select the “Start Sequencing” option to choose the running parameters.
Type in the (1) experiment name, (2) “Select all available” to select all the connected flow cells or use the diagram above to select specific flow cells to run, (3) Check the Flow cell ID and Flow cell type are auto-filled correctly, (4) Copy experiment name and past to the space for sample ID, (5) Select Continue to Kit Selection to move to the next page.
Select the kit SQK-NBD114-96 from the Kit Selection menu. Select Continue to Run Options to choose run parameters.
Set run length to 48 hrs and Minimum read length 200 bp. Let adaptive sampling off. Select Continue to analysis for output setting.
Turn on Basecalling and Barcoding.
Set up Option of Basecalling model in High-accuracy basecalling. Save the setting.
Set up Barcoding options. Turn on Trim barcodes and Mid-read barcode filtering. Save the settings and Continue to output.
Select the output data location, format, and filtering options. Check the box for Raw reads and select POD5. Check the box FASTQ of Basecalled reads. Keep the filter score as the system default. Select “Continue to final review” to proceed.
Review the settings listed in the Run Setup page. Correct any errors. Select “Start” to run the experiment. The system will automatically navigate the Sequencing Overview when sequencing starts.
The system will automatically navigate the Sequencing Overview when sequencing starts.
APPENDIX 4. cDNA END-PREP MASTER MIX PREPARATION
| A | B | C | |
| Component | Volume for 1 reaction | Volume for n+1 reactions | |
| cDNA sample | 20 μl | 20 μl | |
| Nuclease-free water | 30 μl | … μl | |
| Ultra II End-prep reaction buffer | 7 μl | … μl | |
| Ultra II End-prep enzyme mix | 3 μl | … μl | |
| Final total volume | 60 μl | … μl |
APPENDIX 5. EXPECTED OUTCOMES
The DNA or RNA inputs vs the sequencing read yields.
Materials
EQUIPMENT AND MATERIALS
Note
NOTE: If product number is listed, please ensure use of this or equivalent product.
| A | B | |
| Equipment | Mfg / Product # | |
| Oxford Nanopore GridION or MinION Mk1C device or P2 Solo device | Oxford Nanopore Technologies, GRD-CapEx or Oxford Nanopore Technologies, M1CCapEx or PromethION 2 Solo CapEx | |
| Computer monitor (with HDMI port or Display port), mouse and keyboard | Locally sourced | |
| MinKNOW - software equipped already in the GridION and MinION Mk1C device | Oxford Nanopore Technologies | |
| P2 Solo compatible computer with GPU power for real-time basecalling | Locally sourced | |
| Ice bucket with ice | Locally sourced | |
| Qubit fluorometer | ThermoFisher, Q33238 or equivalent | |
| DynaMag-2 magnet | Invitrogen, 12321D or equivalent | |
| DynaMag-96 Side Magnet | Invitrogen, 12331D or equivalent | |
| Hula sample mixer | ThermoFisher, 15920D | |
| Microplate centrifuge | Locally sourced | |
| Timer | Locally sourced | |
| Thermal cycler | Locally sourced | |
| 96-well PCR plate holder | Locally sourced | |
| P1000 pipette and tips | Locally sourced | |
| P200 pipette and tips | Locally sourced | |
| P20 pipette and tips | Locally sourced | |
| P10 pipette and tips | Locally sourced | |
| P10 8-channel pipette | Locally sourced | |
| P300 8-channel pipette | Locally sourced |
| A | B | C | |
| Material | Description | Mfg / Product # | |
| DNA from a sample | Per sample from SOP B-2 (gDNA Preparation) | REDI-NET DNA sample | |
| Eluents from negative control extraction | From SOP B-2 (gDNA Preparation) | REDI-NET negative control | |
| ONT DNA Control Standard (ONT DCS) | 10 ng/μL | Oxford Nanopore, SQK-NBD114.96 | |
| RNA from a sample | Per sample from SOP B-2 (TNA preparation) | REDI-NET RNA sample | |
| MS2 bacteriaphage RNA | 500 ng/μL | Roche, 10165948001 | |
| RNA Storage Solution | Nuclease-free, 1 mM sodium citrate, pH 6.5 buffer | ThermoFisher, AM7000 | |
| Native Barcoding Kit 96 V14 | (Sequencing Library Preparation) | Oxford Nanopore, SQK-NBD114.96 | |
| Turbo DNase and reaction buffer | (cDNA synthesis and amplification) | ThermoFisher, AM2238 | |
| SuperScript IV Reverse Transcriptase | (cDNA synthesis and amplification) | ThermoFisher, 18090010 | |
| RNase inhibitor | For cDNA synthesis and amplification and Host DNA/rRNA depletion | New England Biolabs, M0314L | |
| RLB RT-9N (DNA oligo) | DNA Oligonucleotide, 5’- TTTTTCGTGCGCCGCTTCAACNNNNNNNNN-3’ | Integrated DNA Technologies, Custom DNA oligo, 100 nmole Standard desalting | |
| RLB TSO (chimeric oligo) | Chimeric oligonucleotide, 5’-GCTAATCATTGCTTTTTCGTGCGCCGCTTCAACATrGrGrG-3’ | Integrated DNA Technologies, Custom RNA oligo, 100nmole Standard desalting | |
| RLB PCR primer (DNA oligo) | DNA Oligonucleotide, 5’-TTTTTCGTGCGCCGCTTCA-3’ | Integrated DNA Technologies, Custom DNA oligo, 100nmole Standard desalting | |
| 10 mM dNTP | (cDNA synthesis and amplification) | New England Biolabs, N0447S | |
| Q5 High-Fidelity 2X Master Mix | (cDNA synthesis and amplification) | New England Biolabs, M0492S | |
| Agencourt AMPure XP beads | (Sequencing Library Preparation) | Beckman Coulter, A63881 | |
| NEBNext End repair / dA-tailing Module | (Sequencing Library Preparation) | New England Biolabs, E7546L | |
| NEBNext FFPE Repair Mix | (Sequencing Library Preparation) | New England Biolabs, M6630L | |
| NEB Blunt/TA Ligase Master Mix | (Sequencing Library Preparation) | New England Biolabs, M0367L | |
| NEBNext Quick Ligation Module | (Sequencing Library Preparation) | New England Biolabs, E6056L | |
| R10.4.1 flow cells | Flow cells for sequencing experiment (consumable) | Oxford Nanopore, FLO-MIN114, FLO-PRO114M | |
| UltraPure Bovine Serum Albumin (50mg/ml) | (Sequencing Library Preparation) | ThermoFisher, AM2616 | |
| low DNA binding tubes | 1.5 mL (consumable) | Eppendorf, 022131021 or equivalent | |
| low DNA binding tubes | 2.0 mL (consumable) | Eppendorf, 022431048 or equivalent | |
| PCR tubes | 0.2 mL thin-walled (consumable) | Eppendorf, 951010006 or equivalent | |
| PCR plate | 96 well, low DNA binding, semi-skirted with heat seals (consumable) | Eppendorf, 0030129504 or equivalent | |
| riboPool pan-mammal Kit | For Host rRNA depletion (for TNA form whole blood and buffy coat samples only) | SiTools Biotech, 24 reactions | |
| NEBNext Microbiome DNA enrichment Kit | For Host DNA depletion (for TNA form whole blood and buffy coat samples only) | New England Biolabs, E2612 | |
| RNaseOUT Recombinant Ribonuclease Inhibitor | For Host DNA/rRNA depletion (for TNA form whole blood and buffy coat samples only) | ThermoFisher, 10777019 | |
| BRAND Self-adhesive Plate Sealing Film | Aluminum (consumable) | Fisher Scientific, 13-882-329 | |
| Clear Adhesive Film | For PCR plate sealing | ThermoFisher, 4306311 | |
| Qubit Assay Tubes | For Qubit DNA/RNA measurement (consumable) | Thermo Fisher, Q32856 | |
| Qubit 1X dsDNA HS Assay Kit | (consumable) | ThermoFisher, Q33230 | |
| Qubit RNA HS Assay Kit | (consumable) | ThermoFisher, Q32852 | |
| Nuclease-free water | To prepare ethanol dilutions (consumable) | Locally sourced | |
| Freshly prepared 80% ethanol in nuclease-free water | Prepared from 100% molecular biology grade ethanol (consumable) | Locally sourced | |
| Freshly prepared 70% ethanol in nuclease free water | Prepared from 100% molecular biology grade ethanol (consumable) | Locally sourced | |
| Data sheets | REDI-NET DCS B-4 Testing | REDI-NET Data Portal |
Equipment
Qubit Fluorometer
NAME
Fluorometer
TYPE
Invitrogen
BRAND
Q33238
SKU
LINK
Equipment
DynaMag™-2 Magnet
NAME
Magnet
TYPE
DynaMag™
BRAND
12321D
SKU
LINK
Equipment
Hula mixer
NAME
Mixer
TYPE
Invitrogen
BRAND
15920D
SKU
Any rotator mixer
SPECIFICATIONS
Native Barcoding Kit 96 V14Oxford Nanopore TechnologiesCatalog #SQK-NBD114.96
MS2 bacteriaphage RNARocheCatalog #10165948001
TURBO™ DNase (2 U/µL)Thermo Fisher ScientificCatalog #AM2238
THE RNA Storage SolutionThermo Fisher ScientificCatalog #AM7000
Superscript IV Reverse TranscriptaseLife TechnologiesCatalog #18090050
RNase Inhibitor, Murine - 15,000 unitsNew England BiolabsCatalog #M0314L
Deoxynucleotide (dNTP) Solution MixNew England BiolabsCatalog #N0447S
Q5 High-Fidelity 2X Master Mix - 100 rxnsNew England BiolabsCatalog #M0492S
Agencourt AMPure XP beadsBeckman CoulterCatalog #A63881
NEBNext Ultra II End Repair/dA-Tailing Module - 96 rxnsNew England BiolabsCatalog #E7546L
NEBNext FFPE DNA Repair Mix - 96 rxnsNew England BiolabsCatalog #M6630L
Blunt/TA Ligase Master Mix - 250 rxnsNew England BiolabsCatalog #M0367L
NEBNext Quick Ligation Module - 100 rxnsNew England BiolabsCatalog #E6056L
Nanopore Flow Cell R10.4.1Oxford Nanopore TechnologiesCatalog #FLO-MIN114
DNA LoBind Tubes 2.0 mlEppendorfCatalog #022431048
Eppendorf PCR TubesEppendorfCatalog #951010006
PromethION R10.4.1M flow cellOxford Nanopore TechnologiesCatalog #FLO-PRO114M
96 well LoBind PCR plates Semi-skirtedEppendorfCatalog #0030129504
NEBNext Microbiome DNA Enrichment Kit - 6 rxnsNew England BiolabsCatalog #E2612S
RNaseOUT™ Recombinant Ribonuclease InhibitorThermo Fisher ScientificCatalog #10777019
BRAND™ Self-adhesive Plate Sealing FilmFisher ScientificCatalog #13-882-329
MicroAmp™ Clear Adhesive FilmThermo Fisher ScientificCatalog #4306311
Qubit assay tubesThermo Fisher ScientificCatalog #Q32856
Qubit 1X dsDNA High Sensitivity Assay KitThermo Fisher ScientificCatalog #Q33230
Qubit RNA HS (High Sensitivity) assay Thermo Fisher ScientificCatalog #Q32852
Safety warnings
RISKS AND PERSONAL PROTECTION
Gloves should be worn all the time when handling samples.
Ethics statement
The protocols.io team notes that research involving animals and humans, including their tissues, must be conducted according to internationally-accepted standards and should always have prior approval from an Institutional Ethics Committee or Board.
Before start
BEFORE START
- Must check the DNA and RNA concentrations of your samples of total nucleic acid (TNA).
- If TNAs were prepared from whole blood or buffy coat, follow Appendix 1 for host DNA and rRNA depletion (process either one or both depending on downstream applications), measure the concentration again, and then choose the appropriate approach for sequencing library preparation.
- Select the sequencing approach based on the target of interest: a) For viral targets only, use cDNA to prepare the sequencing library. b) For bacterial targets only, use gDNA. c)If both viral and bacterial targets are of interest, use TNA to prepare the sequencing library.
- gDNA sequencing does not involve any PCR steps and should be handled exclusively in the designated pre-PCR area of the lab.
- The gDNA preparation for TNA sequencing can be handled in the designated pre-PCR area of the lab before being mixed with amplified cDNA.
- Any step involving PCR-amplified cDNA must be handled in the designated post-PCR area of the lab to avoid library contamination.
- The designated post-PCR area of the lab should be cleaned with 1% bleach followed by 70% ethanol before use.
- Use sections gDNA APPROACH, TNA APPROACH, and cDNA APPROACH for gDNA, TNA, and cDNA preparation, respectively, then proceed with the prepared gDNA, TNA or cDNA to Section SEQUENCING LIBRARY PREPARATION.
- The capacity of the sequencing flow cells is limited. The table below shows the maximum allowable DNA and RNA amount and volumes of one sample for each corresponding sequencing approach.
| A | B | C | D | |
| DNA amount (gDNA approach) | Sample volume | Nuclease-free water (μL) | Total volume (μL) | |
| 200 ng, DNA | x | 20-x | 20 |
| A | B | C | D | |
| DNA and RNA amount (TNA approach) | Sample volume | Nuclease-free water (μL) | Total volume (μL) | |
| 40 ng, RNA | x | 20-x | 20 | |
| 200 ng, DNA | x | 10-x | 10 |
| A | B | C | D | |
| RNA amount (cDNA approach) | Sample volume | Nuclease-free water (μL) | Total volume (μL) | |
| 40 ng, RNA | x | 20-x | 20 |
gDNA APPROACH
If the DNA concentration >10 ng/µl , calculate the required volume of 200 ng DNA, then transfer the volume to a new 200 µL PCR tube or a well of a 96-well PCR plate. Adjust the volume with nuclease-free water to a final volume of 20 µL . Directly use a 20 μL sample for the downstream
preparation if the DNA concentration is below 10 ng/μL (for MinION: 22 samples plus 1 positive and 1 negative control; for PromethION: 46 samples plus 1 positive and 1 negative control).
Prepare gDNA from positive control from a ONT DNA Control Standard in 19 µL nuclease-free water in a new 200 µL PCR tube or a well of a 96-well PCR plate. Add 1 µL of ONT DNA Control Standard (DCS) from ONT SQK-NBD114.96 kit to the well containing 19 µL nuclease free water. Mix well by pipetting.
Transfer 20 µL negative control extraction to a new tube or a well of a 96-well PCR plate.
All samples are subjected to section SEQUENCING LIBRARY PREPARATION.
TNA APPROACH
cDNA and gDNA from the same sample are prepared separately, then combined for a single sequencing library preparation.
Use the table in "Before Start" section to prepare 200 ng of DNA in 10 µL of nuclease-free water in a new 200 µL PCR tube or a well of a 96-well PCR plate. If the DNA concentration is below 20 ng/µl , directly transfer10 µL of the sample to the tube or well.
Prepare the gDNA from a positive control using 9 µL of nuclease-free water in a new 200 µL PCR tube or a well of a 96-well PCR plate. Add 1 µL of ONT DCS from ONT SQK-NBD1114.96 kit to the well containing 9 µL nuclease free water. Mix well by pipetting.
Transfer 10 µL of the negative control extraction to a new tube or a well of a 96-well PCR plate.
Prepare double stranded cDNA from RNA following the steps described in sections cDNA APPROACH and cDNA SYNTHESIS AND AMPLIFICATION.
Add 3 µL of final amplified cDNA products from section cDNA APPROACH and 7 µL of nuclease-free water into each well of the 96-well PCR plate containing the corresponding 10 µL of gDNA prepared in prepared in steps 6-8 (including the samples, positive control, and negative control). CAUTION: be careful not to mix materials from different samples or controls.
NOTE: After cDNA amplification and purification (the final products from section cDNA SYNTHESIS AND AMPLIFICATION, the concentration in each sample may range from
50 ng/µL to 300 ng/µL , typically between 80-150 ng/µL . When preparing cDNA and gDNA in a single library preparation reaction, the ideal cDNA quantity per sample is 40-300 ng . For consistency and efficient handling in bulk preparation, a fixed 3 µL of cDNA should generally provide the required amount for sequencing. If the concentration falls outside the expected range, adjust the volumes of cDNA and nuclease-free water accordingly. The total volume of cDNA plus water must not exceed 110 µL .
Subject the 20 µL double-stranded gDNA/cDNA mixture to section SEQUENCING LIBRARY PREPARATION.
cDNA APPROACH
Use the tables in the "Before Start" section to prepare 40 ng of RNA in 20 µL of nuclease-free water in a new 200 µL PCR tube or a well of a 96-well PCR plate. If the RNA concentration is below 2.0 ng/µl , directly transfer 20 µL of the sample to the tube or well.
Prepare 20 ng RNA positive control of MS2 Bacteriophage RNA.
NOTE: MS2 bacteriophage RNA consists of extracted RNA at a concentration of 500 ng/µL or higher in a total volume of 500 µL (the company labeled concentration could be inaccurate). Upon arrival, store the stock below -80 °C . When using the first-time, take 1 µL of RNA stock and mix with 9 µL of nuclease-free water in a 1.5 mL tube to make a 10-fold dilution. Use 1 µL of the 10-fold diluted RNA to measure the concentration using Qubit RNA HS Assay Kit. After the concentration traction measurement, aliquote 50 µL of the stock RNA to 1.5 mL nuclease-free, low-binding tubes and label the concentration on the aliquots for -80 °C storage and future use. Use one 50 µL aliquot to prepare a diluted stock in RNA storage solution at a final concentration of
250 ng/µL . Aliquot the diluted stock into 1.5 mL nuclease-free, low-binding tubes for 2 µL per tube. Store the aliquot diluted stocks at -80 °C and use one aliquot at a time to prevent repeated freeze/thaw.
Preparing the MS2 bacteriophage RNA positive control
Retrieve a tube of the 2 µL MS2 RNA aliquot (250 ng/µL) from the -80 °C freezer.
Add 98 µL of RNA storage solution directly to the aliquot, which contains 2 µL of concentrated bacteriophage MS2 RNA. This will prepare an RNA working solution with a final concentration of 5 ng/µL in 100 µL .
Keep the tube on ice at all times to maintain RNA stability.
In the 96-well sample plate, add 16 µL of nuclease-free water to a designated well.
Add 4 µL of the RNA working solution to the same well, pipetting mix with the 16 µL of nuclease-free water.
The remaining 96 µL of the RNA working solution can be stored at -80 °C for two additional uses, after which any leftover solution should be discarded.
cDNA APPROACH
Transfer 20 µL negative control extraction to a new tube or a well of a 96-well PCR plate.
Use prepared samples, positive control, and negative control for cDNA SYNTHESIS AND AMPLIFICATION (starting at step 17).
cDNA APPROACH
Add 10 µL nuclease-free water to the 10 µL purified double-stranded cDNA purified final product for SEQUENCING LIBRARY PREPARATION.
NOTE: After cDNA SYNTHESIS AND AMPLIFICATION, the concentration in each sample may range from 50 ng/µL to
300 ng/µL , typically between 80-150 ng/µL .
cDNA SYNTHESIS AND AMPLIFICATION
3h 30m
Remove contaminated DNA (~ 30 mins):
BEFORE START:
- Prepare the reactions in the designated pre-PCR area to minimize the risk of cross-contamination.
- Use dedicated pipettes for pre-PCR tasks and use disposable, aerosol-resistant filter tips.
- Thaw total nucleic acid and 10x Turbo DNase Buffer on ice
- Vortex 10x Turbo DNase Buffer briefly, spin down by centrifugation for 5 seconds, and place on ice.
- Place Turbo DNase on ice all the time.
- Set up thermal cycler programs: Set lid temperature at 80 °C . Set up thermal cycler programs:37 °C , 00:20:00 and hold at 4 °C .
25m
Prepare a master mix of 10× Turbo DNase buffer and Turbo DNase following the table below with a 10% overage to account for pipetting variability (N: number of samples plus controls).
| A | B | C | |
| Component | Volume per reaction | Master mix volume (μL) | |
| 10× Turbo DNase Buffer | 3 μL | (3 × N)×1.1= | |
| Turbo DNase (2U/μL) | 1 μL | (1 × N)×1.1= | |
| RNase Inhibitor (40U/μL) | 0.75 μL | (0.75 × N)×1.1= | |
| Nuclease-free water | 5.25 μL | (5.25 × N)×1.1= | |
| Total volume | 10 μL | (10 × N)×1.1= |
Aliquot 10 µL of the prepared master mix into each well of the sample plate outlined in Section cDNA APPROACH, ensuring it is added to wells containing 20 µL of samples or controls.
The final volume per well should be 30 µL . Seal the plate with a self-adhesive plate-sealing film.
Gently mix the samples then centrifuge the sample plate for 00:01:00 .
1m
Incubate the sample for 00:20:00 at 37 °C and hold at 4 °C .
20m
DNA-free RNA purification (~ 30 mins)
Centrifuge the sample plate for 00:01:00 1 min (use the highest available speed of a bench-top plate centrifuge, generally between 500-3000 × g, depending on the model. If the speed is <1000 × g, centrifuge the plate for 00:02:00 ).
3m
Resuspend the AMPure XP beads by vortexing for 00:00:20 .
Carefully remove the plate seal to avoid cross-contamination of the droplets.
Add 30 µL of resuspended AMPure XP beads to the reaction wells in the sample plate and mix by pipetting 5-10 times.
Seal the plate with a new self-adhesive plate-sealing film.
Incubate the sample plate on a rotator mixer at a speed of >1600 rpm for 00:05:00 at Room temperature (If a mixer is unavailable, vortex the plate every 00:02:00 ).
5m
Centrifuge the sample plate for 00:01:00 .
1m
Place the plate on a magnet for a 96-well plate. Keep the plate on the magnet for00:03:00 to 00:05:00 until the supernatants are clear.
5m
While on the magnet, carefully remove the plate seal to avoid cross-contamination of the droplets.
Pipette off the supernatants without touching the bead pellet.
Keep the plate on the magnet and wash the beads with 200 µL of freshly prepared 70% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
Repeat the previous step.
Seal the plate with a new self-adhesive plate-sealing film.
Spin down and place the plate back on the magnet. Remove the seal and pipette off any residual ethanol. Allow to dry for ~30 seconds, but do not dry the pellet to the point of cracking.
Remove the plate from the magnetic rack and remove the seal. Resuspend the pellet in 13 µL nuclease-free water by pipetting for 00:05:00 to 00:10:00 .
15m
Seal the plate with a new self-adhesive plate-sealing film.
Incubate the sample plate on a rotator mixer at a speed >1600 rpm for 00:10:00 at Room temperature (If a mixer is unavailable, vortex the plate every 2 minutes).
10m
Spin down and pellet beads on the magnet until the elute is clear and colorless.
Transfer 11 µL of eluate of each sample to a new 96-well PCR plate. Label the plate as DNA-free RNA with date.
Optional: Measure the RNA quantity using a Qubit fluorometer and Qubit RNA HS Assay Kit.
Seal the plate with a new self-adhesive plate-sealing film.
Use the purified 10 μL DNA-free RNA for the cDNA or TNA sequencing approach00:10:00
Smart-9N First-Strand cDNA synthesis (~ 2.5 hr)
NOTE: Upon the first time use, resuspend the lyophilized RLB RT-9N oligo in nuclease-free water at a molar concentration of 100 μM as stock solution. Store the DNA oligo stock at -20 °C . Also, resuspend the lyophilized RLB TSO oligo in RNA storage solution at a molar concentration of
100 micromolar (µM) as stock solution. Aliquot the RLB TSO oligo stock into 1.5 mL Nuclease-free, low-binding tubes for 3 µL per tube. Store the aliquots at -80 °C .
BEFORE START:
- Prepare the reactions in the designated pre-PCR area to minimize the risk of cross-contamination.
- Use dedicated pipettes for pre-PCR tasks and use disposable, aerosol-resistant filter tips.
- Making
- 2 micromolar (µM) RLB RT-9N DNA oligo: Thaw the RLB RT-9N DNA oligo stock on ice. In a new RNase-free low-binding 1.5 mL tube, combine 3 µL of 100 micromolar (µM) RLB RT-9N DNA oligo stock with 147 µL of nuclease-free water to make a2 micromolar (µM) working solution. Mix the diluted oligo thoroughly by vortexing. The unused 2 micromolar (µM) working solution can be kept at -20 °C for repeated use.
- Making 2 micromolar (µM) RLB TSO oligo: Retrieve a tube of RLB TSO RNA oligo aliquot (3 µL ) from the -80 °C freezer. Add 147 µL of nuclease-free water directly to the tube to prepare the 2 micromolar (µM) RLB TSO oligo working solution. Keep it on ice.
- NOTE: The RLB TSO oligo working solution must be freshly prepared each time.
- Thaw a tube of 10 millimolar (mM) dNTP, along with the 5× SuperScript IV RT buffer and 0.1 Molarity (M) DTT provided with the SuperScript IV reverse transcriptase at room temperature. After thawing, keep the tubes on ice.
- Place SuperScript IV reverse transcriptase and RNase inhibitor on ice.
- Set up thermal cycler: Set lid temperature at 100 °C . Set up two programs as follows,
- 1) RNA denature: 65 °C , 00:05:00 , then hold at 4 °C .
- 2) Template switch RT: 50 °C for01:30:00 , 70 °C for 00:10:00 , then hold at4 °C .
5m
Prepare a master mix of 10 millimolar (mM) dNTP and 2 micromolar (µM) RLB RT 9N oligo following the table below with a 20 % overage to account for pipetting variability (N: number of samples plus controls).
| Component | Volume per reaction | Master mix volume (μL) | |
| 10 mM dNTP | 1 μL | (1 × N)×1.2= | |
| 2 μM RLB RT 9N | 1 μL | (1 × N)×1.2= | |
| Total volume | 2 μL | (2 × N)×1.2= |
Vortex the master mix gently, then spin down.
Aliquot 2 µL master mix into each purified 10 µL DNA-free RNA sample in the plate prepared in Step 40. Mix well the 12 µL reactions by pipetting.
Seal the plate with a new self-adhesive plate-sealing film.
Heat the plate on the thermocycler to 65 °C for 00:05:00 and hold at 4°C.
5m
Prepare a master mix of SuperScript IV reverse transcriptase (SS4RT) following the table below with a 10% overage to account for pipetting variability (N: number of samples plus controls).
| A | B | C | |
| Component | Volume per reaction | Master mix volume (μL) | |
| 5× RT buffer | 4 μL | (4 × N)×1.1= | |
| RNase inhibitor | 1 μL | (1 × N)×1.1= | |
| 2 μM RLB TSO oligo | 1 μL | (1 × N)×1.1= | |
| 0.1M DTT | 1 μL | (1 × N)×1.1= | |
| SuperScript IV reverse transcriptase (SS4RT) | 1 μL | (1 × N)×1.1= | |
| Total volume | 8 μL | (8 × N)×1.1= |
Vortex the master mix gently, then spin down.
Make sure the plate in Step 49 has been cooled at 4 °C . Place the plate On ice .
15m
Carefully remove the plate seal. Aliquot 8 µL of the SS4RT master mix into each reaction well of the plate. Mix thoroughly by pipetting.
Seal the plate with a new self-adhesive plate-sealing film.
Mix gently and centrifuge briefly.
Incubate at 50 °C for 01:30:00 followed by 70 °C for 00:10:00 and then hold at 4 °C .
1h 40m
cDNA amplification (~ 3 hr)
NOTE: For first time use, resuspend the lyophilized RLB PCR DNA oligo in nuclease-free water at a molar concentration of
100 micromolar (µM) as stock solution. Store the DNA oligo stock at -20 °C .
BEFORE START:
- Prepare the reactions in the designated Pre-PCR area to minimize the risk of cross-contamination.
- Use dedicated pipettes for Pre-PCR tasks and use disposable, aerosol-resistant filter tips.
- Always work from clean (pre-PCR) to contaminated (post-PCR) areas, and never backtrack.
- Making 10 micromolar (µM) RLB PCR DNA oligo: Thaw the
- 100 micromolar (µM) oligo stock On ice . In a new RNase-free low-binding 1.5 mL tube, combine 10 µL of oligo stock with 90 µL of nuclease-free water to make a 10 micromolar (µM) working solution. Mix the diluted oligo thoroughly by vortexing. The unused 10 micromolar (µM) working solution can be kept at -20 °C for repeated use.
- Set up thermal cycler: Set lid temperature at100 °C , and set up the cDNA amplification program following the table below.
| Step | Temp (༠C) | Duration | Cycle | |
| Initial denaturation | 95 | 3 mins | 1 | |
| Denaturation | 98 | 15 secs | 27 | |
| Annealing | 62 | 15 secs | ||
| Extension | 65 | 5 mins | ||
| Final extension | 65 | 10 mins | 1 | |
| Storage | 4 | Hold | 1 |
Prepare a master mix for PCR reaction following the table below with a 10 % overage to account for pipetting variability (N: number of samples plus controls).
| Component | Volume per reaction | Master mix volume (μL) | |
| 10 μM RLB PCR oligo | 2 μL | (2 × N)×1.1= | |
| Q5 High-Fidelity 2X Master Mix | 15 μL | (15 × N)×1.1= | |
| Nuclease-free water | 8 μL | (8 × N)×1.1= | |
| Total volume | 25 μL | (25 × N)×1.1= |
Vortex the master mix gently, then spin down.
In a new 96-well PCR plate, aliquot 25 µL of the PCR reaction master mix into the wells designated for each sample.
Add 5 µL of the first-strand cDNA products prepared in Step 56 into each well containing the PCR master mix. Ensure thorough mixing by pipetting gently.
Seal the plate with a new self-adhesive plate-sealing film.
Run the 96-well PCR plate in the thermal cycler using the program for cDNA amplification.
Remaining cDNA products can be stored at -20 °C in case of test failure. Once sequencing is successfully complete, it can be discarded.
STOP POINT: The amplified double-stranded cDNA can be stored at -20 °C before purification.
cDNA SYNTHESIS: Purification of double-stranded cDNA (~ 15 mins)
16m 30s
Purification of amplified double-stranded cDNA (~ 30 mins):
Note
NOTE: Always keep PCR tubes and plates sealed when not in use. Briefly spin down tubes before opening to prevent aerosols. Open tubes and plates gently and use slow pipetting techniques to avoid cross-contamination.
BEFORE START:
- Always work from clean (pre-PCR) to contaminated (post-PCR) areas, and never backtrack.
- Wipe the bench top with 1% bleach, then use 70% ethanol to wipe the bench top again.
- Prepare the reactions in the designated Post-PCR area to minimize the risk of cross-contamination.
- Use dedicated pipettes for Post-PCR tasks and use disposable, aerosol-resistant filter tips.
- Prepare fresh 70% ethanol in nuclease-free water sufficient for your samples. (500 µL per sample).
Centrifuge the sample plate for 00:02:00 (use the highest available speed of a bench-top plate centrifuge, generally between 500-3000 × g, depending on the model. If the speed <1000 × g, centrifuge the plate for 00:03:00 ).
2m
Add 30 µL of resuspended AMPure XP beads to the reaction wells in the sample plate and mix by slow pipetting 5-10 times, changing the tip every time to avoid cross-contamination.
Seal the plate with a new self-adhesive plate-sealing film.
Incubate the sample plate on a rotator mixer at a speed of >1600 rpm for 00:05:00 at Room temperature .(If a mixer is unavailable, vortex the plate every 2 minutes).
5m
Centrifuge the plate for 00:02:00 and pellet the beads on the magnet until the supernatant looks clear.
2m
Keep the plate on the magnet, and pipette off the supernatant. Be careful not to touch the bead pellet.
Keep the plate on the magnet and wash the beads with 200 µL of freshly prepared 70% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
Repeat the previous step X1.
Seal the plate with a new self-adhesive plate-sealing film.
Centrifuge the plate for 00:01:00 and place the plate back on the magnet. Using a pipette, remove any residual ethanol. Allow to dry for ~00:00:30 , but do not dry the pellet to the point of cracking.
1m 30s
Remove the tube from the magnetic rack and resuspend the pellet in 13 µL nuclease-free water by slow pipetting to elute the cDNA.
Seal the plate with a new self-adhesive plate-sealing film.
Incubate on a rotator mixer at speed of>1600 rpm) for 00:05:00 at Room temperature (If a mixer is unavailable, vortex the plate every 2 minutes).
5m
Centrifuge the plate for 00:01:00 and place the plate back on the magnet until the eluate is clear and colorless.
1m
Remove and retain 11 µL of eluate into a new 96-well PCR plate.
Analyze 1 µL of the purified double-stranded cDNA for quantity using Qubit fluorometer and Qubit 1X dsDNA HS Assay Kit.
For the downstream TNA or cDNA sequencing approach, refer to TNA APPROACH or cDNA APPROACH.
Note
STOP POINT: The synthesized double-stranded cDNA can be stored at -20 °C before sequencing.
SEQUENCING LIBRARY PREPARATION
10m
BEFORE START
- When preparing a TNA or cDNA sequencing library with PCR-amplified cDNA, the reactions must be handled in the designated post-PCR area of the lab.
- Briefly spin down the sample plate before opening to prevent aerosols.
- Spin down Ultra II End-prep Enzyme Mix, FFPE DNA Repair Mix and Blunt/TA Ligase Master Mix, then place On ice .
- Thaw the Ultra II End-prep reaction buffer, NEBNext FFPE DNA Repair Buffer, and items from the ONT SQK-NBD 114.96 Kit (Barcode Plate, EDTA, AMPure XP bead) On ice .
- After thawing, Ultra II End-prep reaction buffer and NEBNext FFPE DNA Repair Buffer may form precipitates. If this occurs, equilibrate the reagents to room temperature and vortex before use.
- Prepare fresh 70% ethanol in nuclease-free water (1 mL per sample).
- Prepare fresh 80% ethanol in nuclease-free water (1.5 mL per sample).
- Program a thermal cycler: 20 °C for 00:05:00 and 65 °C for00:05:00 .
- Spin down all reagents briefly before use.
Note
NOTE: Depending on the chosen sequencing approach, the materials subjected to downstream steps at this point should be either gDNA, cDNA, or a mixture of both gDNA and cDNA (called TNA) in 20 µL nuclease-free water.
10m
SEQUENCING LIBRARY PREPARATION: End-prep (~ 50 minutes)
19m 30s
Mix the following reagents to make a master mix for End-prep. When working with multiple samples, prepare a master mix by multiplying the ingredients (except gDNA/cDNA/TNA) with a 20% overage account for pipetting variability (N: number of samples plus controls).
| Component | Volume per reaction | Master mix volume (μL) | |
| Nuclease-free water | 4 μL | (4 × N)×1.2= | |
| Ultra II End-prep reaction buffer | 1.75 μL | (1.75 × N)×1.2= | |
| Ultra II End-prep enzyme mix | 1.5 μL | (1.5 × N)×1.2= | |
| NEBNext FFPE DNA Repair Buffer | 1.75 μL | (1.75 × N)×1.2= | |
| NEBNext FFPE DNA Repair Mix | 1 μL | (1 × N)×1.2= | |
| Final total volume | 10 μL | (10 × N)×1.2= |
Spin down the plate, and remove the plate seal carefully. Aliquot 10 µL of the master mix to each well of a 96-well PCR plate containing 20 µL of samples or controls. Mix 30 µL End-prep reactions gently by pipetting and spin down.
Using a thermal cycler, incubate at 20 °C for 00:05:00 and 65 °C for 00:05:00 .
10m
Resuspend the AMPure XP beads by vortexing.
Add 50 µL of resuspended AMPure XP beads to the end-prep reaction and mix by pipetting (use an 8-channel pipette for reagent transfer of multiple samples).
Incubate on a Hula mixer (a rotator mixer >1600 rpm) for 00:05:00 at Room temperature . (If a mixer is unavailable, vortex the plate every 2 minutes)
5m
SEQUENCING LIBRARY PREPARATION: End-prep (~ 50 minutes)
19m 30s
Seal the plate with a new self-adhesive plate-sealing film.
SEQUENCING LIBRARY PREPARATION: End-prep (~ 50 minutes)
19m 30s
Centrifuge the plate for00:02:00 and pellet the beads on the magnet until the supernatant looks clear. (DynaMag-2 for 1.5mL tubes and DynaMag-96 for a PCR plate) and pipette off the supernatant without disturbing the beads
2m
Remove the seal. Keep the sample on the magnet and wash the beads with 200 µL of freshly prepared 70% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
Repeat the previous step X1.
SEQUENCING LIBRARY PREPARATION: End-prep (~ 50 minutes)
19m 30s
Seal the plate with a new self-adhesive plate-sealing film.
SEQUENCING LIBRARY PREPARATION: End-prep (~ 50 minutes)
19m 30s
Spin down and place the tube back on the magnet. Using a pipette, remove any residual ethanol. Allow to dry for ~00:00:30 , but do not dry the pellet to the point of cracking.
30s
Remove the tube from the magnetic rack and resuspend the pellet in 12 µL nuclease-free water by pipetting.
2m
SEQUENCING LIBRARY PREPARATION: End-prep (~ 50 minutes)
19m 30s
Seal the plate with a new self-adhesive plate-sealing film.
Incubate for 00:02:00 at Room temperature .
SEQUENCING LIBRARY PREPARATION: End-prep (~ 50 minutes)
19m 30s
Spin down and place the tube back on the magnet until the eluate is clear and colorless.
Remove the seal and transfer 11 µL of eluate into a cleanwells on the same plate, maintaining the original column/row pattern while skipping previously used columns and rows. (For example, if columns 1-6 were used, transfer the elutes in parallel to columns 7-12).
SEQUENCING LIBRARY PREPARATION: End-prep (~ 50 minutes)
19m 30s
Directly use the product to barcode ligation or seal the plate with a new self-adhesive plate-sealing film.
STOP POINT: The purified End-prep reactions can be stored at -20 °C before barcode ligation.
SEQUENCING LIBRARY PREPARATION: Barcode ligation (~ 25 minutes)
The table below lists reagents for one barcoding reaction. Add the reagents to the End-prepped sample following the listed order. Carefully check the plate layout and recode numbers for the barcodes when transferring. (Optional: use multi-channel pipette for barcode transfer when working with 24 or 48 samples). Seal the used barcodes with a cut-off piece of a self-adhesive aluminum foil.
| A | B | |
| Component | Volume | |
| End-prepped DNA | 10 μl | |
| Native Barcode (pick one form Native Barcoding Expansion 1-96) | 2 μl | |
| Blunt/TA Ligase Master Mix | 12 μl | |
| Final total volume | 24 μl |
Mix gently by pipetting 10-15 times, seal, and spin down the plate.
Incubate the reaction for 00:20:00 at Room temperature .
20m
Spin down the plate and remove the plate seal. Add 3 µL of EDTA to each well and mix thoroughly by pipetting and spin down briefly.
Note
At this point, samples should be individually barcoded and ready to be subjected to pooling.
Seal the plate and spin down.
SEQUENCING LIBRARY PREPARATION: Library pooling for multiplex sequencing
Resuspend the AMPure XP beads by vortexing.
Use the table below to pool the barcoded samples in a new 1.5 mL low DNA binding tube depending on the flow cell that is going to be used. Add resuspended AMPure XP beads to the pooled library and mix by pipetting.
| A | B | C | |
| MinION (R10.4.1) | PromethION (R10.4.1) | ||
| Each barcoded sample (μL) | 12 | 12 | |
| Number of samples in a pool | 24 | 48 | |
| Blunt/TA Ligase Master Mix (NEB M0367L, ready-to-use) | 288 | 576 | |
| Total volume of a pool (μL) AMPure XP beads for purification (μL, 0.5x of the pooled library volume) | 144 | 288 |
Incubate on a Hula mixer (or a rotator mixer) for 00:10:00 at Room temperature .
10m
Spin down the sample and pellet on a magnet. Keep the tube on the magnet for 00:05:00 , and using a pipette, discard the supernatant.
5m
Keep the tube on the magnet and wash the beads with 700 µL of freshly prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
Repeat the previous step X1.
Spin down and place the tube back on the magnet. Using a pipette, remove any residual ethanol. Allow to dry for ~00:00:30 , but do not dry the pellet to the point of cracking.
30s
Remove the tube from the magnetic rack and resuspend the pellet in 32 µL nuclease-free water. Incubate for 00:10:00 at 37 °C temperature,agitating the sample for 10 seconds every 2 minutes. Alternatively, if available, mix the sample on a thermomixer at 37°C at ≥1600 rpm.
10m
Spin down and pellet the beads on a magnet until the eluate is clear and colorless.
Remove and retain 31 µL of eluate into a clean 1.5 mL low DNA binding tube.
SEQUENCING LIBRARY PREPARATION: Adapter ligation (~ 45 minutes)
30m
BEFORE STARTING: Thaw Short Fragment Buffer (SFB), Elution Buffer (EB), and NEBNext Quick Ligation Reaction Buffer (5×) at Room temperature , mix by vortexing, spin down, and place On ice . Check that the contents or each tube are clear of any precipitate. Spin down the T4 Ligase and the Native Adapter (NA), and place On ice .
Taking the pooled and barcoded DNA, perform adapter ligation as follows, mix by flicking the tube between each sequential addition.
| A | B | |
| Pooled barcoded sample | 30 μl | |
| Native Adapter (NA) | 5 μl | |
| NEBNext Quick Ligation Reaction Buffer (5×) | 10 μl | |
| Quick T4 DNA Ligase | 5 μl | |
| Final total volume | 50 μl |
Mix gently by flicking the tube, and spin down.
Incubate the reaction for 00:10:00 at Room temperature .
10m
Resuspend the AMPure XP beads by vortexing.
Add 50 µL of resuspended AMPure XP beads to the reaction and mix by pipetting.
Incubate on a Hula mixer (rotator mixer) for 00:10:00 at Room temperature .
10m
Place on the magnetic rack, allow beads to pellet and using a pipette, discard the supernatant.
Add 125 µL of the Short Fragment Buffer (SFB) to the beads. Close the tube lid and resuspend the beads by flicking the tube. Return the tube to the magnetic rack, allow beads to pellet, and using a pipette, discard the supernatant.
Repeat the previous step X1.
Spin down and place the tube back on the magnet. Using a pipette, remove any residual supernatant.
Remove the tube from the magnetic rack and resuspend the pellet in Elution Buffer (EB), depending on the flow cell to be used.
| A | B | C | |
| MinION (R10.4.1) | PromethION (R10.4.1) | ||
| EB (μL) | 13 | 33 |
Incubate on at 37 °C for 00:10:00 at, agitate the sample for 10s every 2 min. Alternatively, if available, mix the sample on a thermomixer at 37°C at ≥1600 rpm.
10m
Pellet beads on magnet until the eluate is clear and colorless.
Remove and retain 13 µL (MinION) or 33 µL (PromethION) of eluate into a clean 1.5 mL low DNA binding tube.
Quantify 1 µL of eluted sample using a Qubit fluorometer and Qubit 1X dsDNA HS Assay Kit.
The recovery of the library and the expected molar concentration for loading are listed in the table below.
| A | B | C | |
| MinION (R10.4.1) | PromethION (R10.4.1) | ||
| Library volume | 12 | 32 | |
| Expected recovery quantity (ng) | 100-400 | 200-1000 | |
| Expected library molar conc. (fmol) | 30 | 100 | |
| Recommended library loading upper limit | 1000 ng total (83 ng/μL) | 4000 ng total (125 ng/μL) |
Put the library On ice until ready to load or store the library at -20 °C for future sequencing.
Priming and loading the SpotON Flow Cell
Check the number of pores in your flow cell.
Note
NOTE: before starting the flow cell pore checking, check the hardware following the manufacturer's guidance.
If using a P2 Solo device, connect the P2 Solo with the wire included in the P2 Solo package to a GridION or a compatible computer with MinKNOW software via the USB-C port and turn on.
Turn on GridION (or MinION Mk1C) device. Make sure all the connections for the display, mouse, keyboard, and internet are ready.
Get a MinION or a PromethION flow cell from the refrigerator.
Double-click the MinKNOW icon shown on the desktop to initiate the program.
Use Oxford Nanopore Community username and password to login.
Select the device shown on the screen.
Open the lid of the sequencer and insert the flow cells under the clips, press down the flow cell to ensure good thermal and electrical contact.
The Sequencing Overview tab should show the flow cell not checked in each position in use.
Navigate to the Start tab and select Flow Cell Check.
Check that the sequencer assigns the correct flow cell type: FLO-MIN114 for MinION and FLO-PRO114M for PromethION. If not, select the correct flow cell type from the drop-down list.
Click Start to begin the flow cell check.
Record the port number and date of checking on the original package of the flow cell. The flow cell with less than 800 (MinION)/5000(PromethION) pores should not be used for the sequencing. If the flow cell is not expired (12 weeks shelf life), contact Oxford Nanopore Technologies to claim a replacement.
If the flow cell is going to be used immediately, keep it on the GridION or MinION Mk1C sequencer for priming. Otherwise put the flow cell back to the original pouch, store at 4 °C for next day use. The opened flow cell should be used within one week.
Priming and loading the SpotON Flow Cell: Flow cell priming
BEFORE STARTING:
Thaw the Sequencing Buffer (SB), Library Beads (LIB), Flow Cell Tether (FCT) and one tube of Flow Cell Flush (FCF) at TemperatureRoom temperature . Mix SB by tapping or pipetting (DO NOT Vortex) and vortex the other tubes. Spin down tubes at TemperatureRoom temperature .
Check the air bubble of priming pore.
Open the sequencer lid and insert the pore checked flow cell.
Slide the priming port cover (MinION) or inlet port cover (PromethION) clockwise to open the port.
Note
NOTE: Please see Appendix 2 for the positions of the flow cell ports.
Check for a small air bubble under the cover. Draw back a small volume (20-30 µL ) to remove any bubbles:
Set a P1000 pipette to 200 µL . Insert the tip into the priming port. Turn the volume adjustment wheel counter-clockwise until the dial shows 220-230 µL or until you can see a small buffer volume entering the pipette tip.
Note
IMPORTANT: Take care when drawing back the buffer from the flow cell. Do not remove more than 20-30 µL , and make sure that the array of pores is always covered by the buffer. Introducing air bubbles into the array can irreversibly damage pores.
Prepare the flow cell priming mix and prime flow cells.
Using a 2.0 mL low DNA binding tube, prepare flow cell priming mix with components as follows, mix by inverting the tube and pipetting.
| A | B | C | |
| Component | MinION | PromethION | |
| Bovine Serum Albumin (BSA) (50 mg/ml) | 5 μl | - | |
| Flow Cell Tether (FCT) | 30 μl | 30 μl | |
| Flow Cell Flush (FCF) | 1170 μl | 1170 μl | |
| Final total volume | 1205 μl | 1200 μl |
Load 800 µL (MinION) or 500 µL (PromethION) of the priming mix into each flow cell via the priming/inlet port, avoiding the introduction of air bubbles. Wait for 00:05:00 .
5m
Prepare the library for loading.
Note
IMPORTANT: The Library Beads (LIB) tube contains a suspension of beads. These beads settle very quickly. It is vital that they are mixed immediately before use.
Thoroughly mix the contents of the Library Beads (LIB) by pipetting.
In a new tube, prepare each library for loading as follows:
| A | B | C | |
| Component | MinION | PromethION | |
| Sequencing Buffer (SB) | 37.5 μl | 100 μl | |
| Library Beads (LIB) | 25.5 μl | 68 μl | |
| DNA library | 12 μl | 32 μl | |
| Final total volume | 75 μl | 200 μl |
Complete the flow cell priming.
(MinION) Gently lift the SpotON sample port cover to make the SpotON sample port accessible.
Load 200 µL (MinION) or 500 µL (PromethION) of the priming mix into the flow cell via the priming/inlet port, avoiding the introduction of air bubbles.
Loading samples.
Mix the prepared library gently by pipetting up and down just prior to loading.
Add the entire volume of the library loading mix via the SpotON sample port (MinION) or inlet port (Note: PromethION which has only one port for both priming and sample loading) in a dropwise fashion. Ensure each drop flows into the port before adding the next drop.
(MinION) Gently replace the SpotON/inlet port cover, making sure the bung enters the SpotON port, close the priming port.
Press the light shield included in the flow cell poach around the SpotON/inlet port cover firmly to block the light from the pore window.
Close the lid of the sequencer.
Priming and loading the SpotON Flow Cell: Data acquisition and basecalling
Note
NOTE: A series of snapshots showing the way to MinKNOW is listed in Appendix 3.
Double-click the MinKNOW icon displayed on the desktop to initiate the program.
Use Oxford Nanopore Community username and password to login or continue as Guest.
Select the device shown on the screen.
Go to the Start tab, and click the Start Sequencing option to choose the running parameters.
Type in the Experiment Name use scheme as shown below, [YYYY_MM_DD_Approach(gDNA or TNA)_Sample type
Example: 2024_11_25_cDNA_Water
Approaches: gDNA, cDNA, TNA
Sample type names: Soil, Water, Tick, Leech, Feces, Blood, Swab, Filth Fly
Type in Sample ID (same as experiment name)
Choose flow cell FLO-MIN114 for MinION and FLO-PRO114M for PromethION from the drop-down menu.
Use Select all available to select all the connected flow cells or use the diagram above to select specific flow cells to run.
Click Continue to Kit Selection to move to the next page.
Click the kit SQK-NBD114-96 from the Kit Selection menu.
Click Continue to Run Options to choose run parameters.
Set run length to 48:00:00 and minimum read length 200 bp. Leave adaptive sampling unchecked.
2d
Click Continue to Analysis to choose basecalling and Barcoding parameters.
In the Basecalling options, checkup the basecalling with configuration: High accuracy basecalling.
In the Barcoding options, turn on the Trim barcodes and Mid-read barcoding filtering.
Do not turn on the Alignment option.
Click Continue to output to the next page.
Select the output data location, format, and filtering options. Check up the box for Raw reads in POD5 format and Basecalled reads in FASTQ format. Keep the filter score as the system default.
Click Continue to final review to proceed.
Review the settings listed in the Run Setup page. Correct any errors. Select Start to run the experiment.
The system will automatically navigate the Sequencing Overview when sequencing starts.
48 hrs later, check the sequencing data. Use 1 mL pipette to remove 1 mL waste solution in the waste channel via waste port 1 (see Appendix 2 under Guidelines & Warnings tab). Remove the flow cells on the device, put it back in the original package, and turn off the device.
APPENDIX 1. HOST DNA & rRNA DEPLETION for TNA from WHOLE BLOOD & BUFFY COAT
1h 29m 5s
Note
IMPORTANT: Host-depletion is required for TNA from whole blood and buffy coat only, but NOT for other blood sample types including plasma, serum and dried blood spot on FTA cards.
Prepare NEBNext Microbiome DNA Enrichment Kit.
When using the kit for the first time, thaw 5X NEBNext Bind/wash Buffer (48ml bottle) On ice . After defrosting, aliquot the buffer into six 1.5 mL tubes, 1 mL for each tube, then aliquot the rest of the 42 mL buffer into fourteen 15 mL tubes, 3 mL for each tube.
Note
All tubes should be stored at-20 °C before use.
Prepare MBD2-Fc Protein and Magnetic Beads in Microbiome DNA Enrichment Kit for host DNA depletion.
Place one tube of 3 mL 5X Bind/wash buffer, NEBNext Protein A Magnetic Beads, and NEBNext MBD2-Fc Protein On ice .
Add 12 mL Nuclease-free water into the 3 mL 5X Bind/wash buffer in 15mL tube to make 1X Bind/wash buffer and relabel the tube as 1X buffer.
Resuspend NEBNext Protein A Magnetic Beads by gently pipetting the slurry up and down until the suspension is homogeneous; avoid bubbling.
Note
Do not vortex.
In one 1.5 mL tube, add 8 µL of MBD2-Fc protein and 80 µL of Protein A Magnetic Beads. Mix by pipetting up and down (avoid bubbling) until the beads are completely homogeneous, at least 5-10 times.
Note
One mix is used for one sample. If preparing the MBD2-Fc protein and Protein A Magnetic Beads mix for multiple samples, scale up by the number of samples.
Mix the beads in a Hula mixer for 00:03:00 at Room temperature .
3m
Spin the tube for 00:00:05 and use pipetting to resuspend the bottom beads if any. If the mix is prepared for multiple samples, aliquot 80 µL mix to 1.5 mL tubes.
5s
Briefly spin the tube and place in the magnetic rack for 2–5 minutes or until the beads have collected to the wall of the tube and the solution is clear.
Carefully remove the supernatant with a pipette without disturbing the beads.
Add 500 µL of 1X Bind/wash Buffer (kept On ice ) to the tube to wash the beads. Pipette up and down until the beads are completely homogeneous, at least 5-10 times.
Mix the beads in a Hula mixer for 00:03:00 atRoom temperature .
3m
Briefly spin the tube and place on the magnetic rack for 2–5 minutes or until the beads have collected to the wall of the tube and the solution is clear.
Carefully remove the supernatant with a pipette without disturbing the beads.
Repeat steps 119.9 to 119.12 one time.
Remove the tube from the rack and add 80 µL of 1X Bind/wash Buffer (kept On ice ) to resuspend the beads.
The MBD2-Fc-bound magnetic beads now are ready to use for host DNA depletion and stable for up to 7 days at 4 °C .
Prepare riboPOOL Pan-Mammal Kit.
Upon arrival, store the reagents in the kit as instructed on the bottles.
When using the kit for the first time, centrifuge the riboPOOL probe tube (RP) at 11000 x g, 00:00:30 before opening. Add 30 µL kit provided nuclease-free water and vortex well to resuspend the riboPOOL probe. Aliquot the 5 µL RP in 1.5 mL tubes after resuspension. Store RP aliquots at -20 °C . Avoid freeze-thaw cycles.
30s
Host rRNA depletion
Before starting, set up 2 programs on a thermocycler.
- 68 °C , 00:10:00 , then slowly cool down to 37 °C at a speed 3 °C /min (0.05°C/sec); when reached, keep the temperature at 37 °C .
- 37 °C , 00:15:00 , then 50 °C , 00:05:00 .
30m
Transfer TNA containing around 500 ng RNA to a 200 µL PCR tube, adjust volume with nuclease-free water to 13.7 µL . For example, if the RNA concentration of the TNA sample is 100 ng/µl , transfer 5 µL of the sample to a 200 µL PCR tube and add 8.7 µL nuclease-free water. When the RNA concentration is lower than 36.5 µL use 13.7 µL sample without volume adjustment.
Volume (Probe Hybridization: add reagents as the following table for one sample. For processing multiple samples, make a master mix with a 10% overage.
| A | B | |
| Component | Volume (μL) | |
| TNA | 13.7 | |
| RP | 0.3 | |
| 4X Hybridization Buffer (HB) | 5 | |
| RNaseOUT (40U/ μL) | 1 | |
| Total | 20 |
Mix by pipetting, then spin down briefly.
Place the tube in the thermocycler and run the first program 68 °C cool down to 37 °C set at step 115.1.
Prepare streptavidin-coated magnetic beads (SMB): Vortex SMB beads at medium speed. Transfer 45 µL bead suspension per sample to a new 1.5mL tube. For multiple sample processing, calculate the amount of SMB needed by multiply 45 with the sample number.
Briefly spin down, place the tube on a magnetic rack and wait for 00:01:00 .
1m
Aspirate with a pipette and discard all supernatant.
Add 40 µL Depletion Buffer per sample (i.e. 240 µL for 6 samples, 480 µL for 12 samples) and tap the tube to resuspend beads.
Repeat steps 121.7 to 121.9. Beads are now resuspended in Depletion Buffer.
Move the tube in the thermocycler at step 115.5 to a PCR tube rack.
Add 40 µL beads from step 121.5 to the PCR tube and mix by pipetting.
Put the tube back into the thermocycler for incubation at 37 °C for 00:15:00 , then 55 °C for 00:05:00 .
20m
After incubation, briefly spin down droplets.
Transfer 60 µL sample-bead mix in the PCR tube to a new1.5 mL tube.
Place the 1.5 mL tube on the magnet rack for 00:02:00 then carefully transfer the 60 µL supernatant to a new 1.5 mL tube. The supernatant now contains host rRNA-depleted TNA. Keep the supernatant On ice for the next section of Host DNA depletion.
2m
Host DNA depletion.
Add 60 µL host rRNA depleted sample from the previous step (121.16) to a tube containing 80 µL MBD2-Fc-bound magnetic beads prepared at step 119.14.
Note
NOTE: make sure the MBD2-Fc-bound magnetic beads were prepared within 7 days.
Add 35 µL 5X Bind/wash buffer from the 1 mL aliquot prepared at step 118.1.
Mix the sample in the Hula mixer for 00:15:00 at Room temperature .
15m
Briefly spin the tube and place it on the magnetic rack for 00:05:00 until the beads have collected to the wall of the tube and the solution is clear.
5m
Carefully transfer 175 µL supernatant with a pipette, without disturbing the beads to a clean 1.5 mL microcentrifuge tube. The sample at this stage is host rRNA and DNA depleted in buffer. Store this sample at -20 °C or proceed directly to the sample cleanup described in steps 123 - 123.16
AMPure XP Bead Cleanup.
Vortex AMPure XP Beads to resuspend.
Add 175 µL resuspended AMPure XP beads to the sample at step 122.5.
Mix well by pipetting up and down at least 10 times.
Note
Be careful to expel all the liquid from the tip during the last mix.
Incubate samples for at least 00:05:00 at Room temperature .
5m
Quickly spin the sample, then place the tube on the magnet rack to separate the beads from the supernatant.
After 2-5 minutes (or when the solution is clear), using a pipette, remove and discard the supernatant without disturbing the beads .
Add 400 µL of freshly prepared 80% ethanol to the tube on a magnetic rack. Incubate at Room temperature for 00:00:30 , then with a pipette, carefully remove and discard the 80% ethanol without disturbing the beads.
30s
Repeat the previous step X1.
Briefly spin the tube, place it back on the magnetic stand and remove traces of ethanol with a p10 pipette tip.
Air dry the beads for 00:02:00 while the tube is on the magnetic rack with the lid open.
2m
Add 30 µL of nuclease-free water to the beads.
Briefly spin the sample and mix well by pipetting up and down 10 times.
Incubate for 00:02:00 at Room temperature .
2m
Briefly spin the sample, then place the tube on the magnetic rack for 2-5 minutes (or when the solution is clear).
Transfer the supernatant to a new 1.5 mL tube.
Use 1 µL for DNA/ RNA concentration measurement. Refer to REDI-NET SOP B-2 for the method of DNA/RNA quantification and storage.
Protocol references