Jun 03, 2025

Public workspaceREDI-NET FF-4 FILTH FLY TESTING V.2

DOI
dx.doi.org/10.17504/protocols.io.e6nvw17o2lmk/v2
  • 1REDI-NET Consortium
  • Remote Emerging Disease Intelligence - NETwork (REDI-NET)
REDI-NET Consortium
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DOI: dx.doi.org/10.17504/protocols.io.e6nvw17o2lmk/v2
Protocol Citation: REDI-NET Consortium 2025. REDI-NET FF-4 FILTH FLY TESTING. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvw17o2lmk/v2Version 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: September 11, 2024
Last Modified: June 03, 2025
Protocol Integer ID: 138656
Keywords: gDNA PREPARATION, TNA PREPARATION, cDNA PREPARATION, Purification of double-stranded cDNA, SEQUENCING LIBRARY PREPARATION
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
The overarching aim of the REDI-NET is to develop a collaborative laboratory network between domestic and international partnering institutions to address disease surveillance needs in order to effectively detect, predict and contain potentially emergent zoonosis. This SOP provides guidance on testing of filth fly samples and their purified nucleic acid to preserve their integrity for downstream sequencing library preparation.
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 filth fly 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:

  1. RNases are very stable and difficult to inactivate and only minute amounts are sufficient to destroy RNA.
  2. Care should be taken to avoid inadvertently introducing RNases into the samples during or after the purification procedure.
  3. 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 TemperatureOn 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

APPENDICES

APPENDIX 1. FLOW CELL

MinION flow cell:


PromethION flow cell:



APPENDIX 2. 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 3. cDNA END-PREP MASTER MIX PREPARATION
ABC
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 4. EXPECTED OUTCOMES

The DNA or RNA inputs vs the sequencing read yields.






Materials
Note
NOTE: If product number is listed, please ensure use of this or equivalent product.

AB
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 basecallingLocally 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
ABC
Material Description Mfg / Product #
DNA from a sample Per sample from SOP FF-2 (gDNA Preparation) REDI-NET DNA sample
Eluents from negative control extraction From SOP FF-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 FF-2 (TNA preparation) REDI-NET RNA sample
MS2 bacteriaphage RNA 500 ng/μLRoche, 10165948001
RNA Storage SolutionNuclease-free, 1 mM sodium citrate, pH 6.5 bufferThermoFisher, 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 inhibitorFor 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 FF-4 Testing REDI-NET Data Portal

Equipment
Qubit Fluorometer
NAME
Fluorometer
TYPE
Invitrogen
BRAND
Q33238
SKU
https://www.thermofisher.com/order/catalog/product/Q33238#/Q33238
LINK

Equipment
DynaMag™-2 Magnet
NAME
Magnet
TYPE
DynaMag™
BRAND
12321D
SKU
https://www.thermofisher.com/order/catalog/product/12321D
LINK

Equipment
Hula mixer
NAME
Mixer
TYPE
Invitrogen
BRAND
15920D
SKU
Any rotator mixer
SPECIFICATIONS

ReagentNative Barcoding Kit 96 V14Oxford Nanopore TechnologiesCatalog #SQK-NBD114.96

ReagentMS2 bacteriaphage RNARocheCatalog #10165948001

ReagentTURBO™ DNase (2 U/µL)Thermo Fisher ScientificCatalog #AM2238

ReagentTHE RNA Storage SolutionThermo Fisher ScientificCatalog #AM7000

ReagentSuperscript IV Reverse TranscriptaseLife TechnologiesCatalog #18090050

ReagentRNase Inhibitor, Murine - 15,000 unitsNew England BiolabsCatalog #M0314L

ReagentDeoxynucleotide (dNTP) Solution MixNew England BiolabsCatalog #N0447S

ReagentQ5 High-Fidelity 2X Master Mix - 100 rxnsNew England BiolabsCatalog #M0492S

ReagentAgencourt AMPure XP beadsBeckman CoulterCatalog #A63881

ReagentNEBNext Ultra II End Repair/dA-Tailing Module - 96 rxnsNew England BiolabsCatalog #E7546L

ReagentNEBNext FFPE DNA Repair Mix - 96 rxnsNew England BiolabsCatalog #M6630L

ReagentBlunt/TA Ligase Master Mix - 250 rxnsNew England BiolabsCatalog #M0367L

ReagentNEBNext Quick Ligation Module - 100 rxnsNew England BiolabsCatalog #E6056L

ReagentNanopore Flow Cell R10.4.1Oxford Nanopore TechnologiesCatalog #FLO-MIN114

ReagentDNA LoBind Tubes 2.0 mlEppendorfCatalog #022431048

ReagentEppendorf PCR TubesEppendorfCatalog #951010006

ReagentPromethION R10.4.1M flow cellOxford Nanopore TechnologiesCatalog #FLO-PRO114M

Reagent96 well LoBind PCR plates Semi-skirtedEppendorfCatalog #0030129504

ReagentNEBNext Microbiome DNA Enrichment Kit - 6 rxnsNew England BiolabsCatalog #E2612S

ReagentRNaseOUT™ Recombinant Ribonuclease InhibitorThermo Fisher ScientificCatalog #10777019

ReagentBRAND™ Self-adhesive Plate Sealing FilmFisher ScientificCatalog #13-882-329

ReagentMicroAmp™ Clear Adhesive FilmThermo Fisher ScientificCatalog #4306311

ReagentQubit assay tubesThermo Fisher ScientificCatalog #Q32856

ReagentQubit 1X dsDNA High Sensitivity Assay KitThermo Fisher ScientificCatalog #Q33230

ReagentQubit 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.
Before start
BEFORE START

  1. Must check the DNA and RNA concentrations of your samples of total nucleic acid (TNA).
  2. 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.
  3. 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.
  4. gDNA sequencing does not involve any PCR steps and should be handled exclusively in the designated pre-PCR area of the lab.
  5. The gDNA preparation for TNA sequencing can be handled in the designated pre-PCR area of the lab before being mixed with amplified cDNA.
  6. Any step involving PCR-amplified cDNA must be handled in the designated post-PCR area of the lab to avoid library contamination.
  7. The designated post-PCR area of the lab should be cleaned with 1% bleach followed by 70% ethanol before use.
  8. Use sections gDNA APPROACH, TNA APPROACH, and cDNA APPROACH for gDNA, TNA, and cDNA preparation, respectively, then subject the prepared gDNA, TNA and/or cDNA to Section SEQUENCING LIBRARY PREPARATION.
  9. The capacity of the sequencing flow cells is limited. Refer to the table below for the maximum allowable DNA and RNA quantities and volumes of a sample for each corresponding sequencing approach.
ABCD
DNA amount (gDNA approach) Sample volume Nuclease-free water (μL) Total volume (μL)
200 ng, DNAx20-x20
ABCD
DNA and RNA amount (TNA approach)Sample volume Nuclease-free water (μL) Total volume (μL)
40 ng, RNAx20-x20
200 ng, DNAx10-x10
ABCD
RNA amount (cDNA approach)Sample volumeNuclease-free water (μL)Total volume (μL)
40 ng, RNA x20-x20
gDNA APPROACH
gDNA APPROACH
If the DNA concentration >Amount10 ng/µl , calculate the required volume of Amount200 ng DNA, then transfer the volume to a new Amount200 µL PCR tube or a well of a 96-well PCR plate. Adjust the volume with nuclease-free water to a final volume of Amount20 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).
Pipetting
Prepare gDNA from positive control from a ONT DNA Control Standard in Amount19 µL nuclease-free water in a new Amount200 µL PCR tube or a well of a 96-well PCR plate. Add Amount1 µL of ONT DNA Control Standard (DCS) from ONT SQK-NBD114.96 kit to the well containing Amount19 µL nuclease free water. Mix well by pipetting.
Pipetting
Transfer Amount20 µL negative control extraction to a new tube or a well of a 96-well PCR plate.
Pipetting
All samples are subjected to section SEQUENCING LIBRARY PREPARATION.
TNA APPROACH
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 Amount200 ng of DNA in Amount10 µL of nuclease-free water in a new Amount200 µL PCR tube or a well of a 96-well PCR plate. If the DNA concentration is below Amount20 ng/µl , directly transferAmount10 µL of the sample to the tube or well.
Pipetting
Prepare the gDNA from a positive control using Amount9 µL of nuclease-free water in a new Amount200 µL PCR tube or a well of a 96-well PCR plate. Add Amount1 µL of ONT DCS from ONT SQK-NBD1114.96 kit to the well containing Amount9 µL nuclease free water. Mix well by pipetting.
Pipetting
Transfer Amount10 µL of the negative control extraction to a new tube or a well of a 96-well PCR plate.
Pipetting
Prepare double stranded cDNA from RNA following the steps described in sections cDNA APPROACH and cDNA SYNTHESIS AND AMPLIFICATION.
Add Amount3 µL of final amplified cDNA products from section cDNA APPROACH and Amount7 µL of nuclease-free water into each well of the 96-well PCR plate containing the corresponding Amount10 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
Concentration50 ng/µL to Concentration300 ng/µL , typically between Concentration80-150 ng/µL . When preparing cDNA and gDNA in a single library preparation reaction, the ideal cDNA quantity per sample is Amount40-300 ng . For consistency and efficient handling in bulk preparation, a fixed Amount3 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
Pipetting
Mix
Subject the Amount20 µL double-stranded gDNA/cDNA mixture to section SEQUENCING LIBRARY PREPARATION.
cDNA APPROACH
cDNA APPROACH
Use the tables in the "Before Start" section to prepare Amount40 ng of RNA in Amount20 µL of nuclease-free water in a new Amount200 µL PCR tube or a well of a 96-well PCR plate. If the RNA concentration is below Amount2.0 ng/µl , directly transfer Amount20 µL of the sample to the tube or well.
Pipetting
Prepare Amount20 ng RNA positive control of MS2 Bacteriophage RNA.

NOTE: MS2 bacteriophage RNA consists of extracted RNA at a concentration of Concentration500 ng/µL or higher in a total volume of Amount500 µL (the company labeled concentration could be inaccurate). Upon arrival, store the stock below Temperature-80 °C . When using the first-time, take Amount1 µL of RNA stock and mix with Amount9 µL of nuclease-free water in a 1.5 mL tube to make a 10-fold dilution. Use Amount1 µL of the 10-fold diluted RNA to measure the concentration using Qubit RNA HS Assay Kit. After the concentration traction measurement, aliquote Amount50 µL of the stock RNA to 1.5 mL nuclease-free, low-binding tubes and label the concentration on the aliquots for Temperature-80 °C storage and future use. Use one Amount50 µL aliquot to prepare a diluted stock in RNA storage solution at a final concentration of
Concentration250 ng/µL . Aliquot the diluted stock into 1.5 mL nuclease-free, low-binding tubes for Amount2 µL per tube. Store the aliquot diluted stocks at Temperature-80 °C and use one aliquot at a time to prevent repeated freeze/thaw.
Pipetting
Preparing the MS2 bacteriophage RNA positive control
Preparing the MS2 bacteriophage RNA positive control
Retrieve a tube of the Amount2 µL MS2 RNA aliquot (250 ng/µL) from the Temperature-80 °C freezer.
Add Amount98 µL of RNA storage solution directly to the aliquot, which contains Amount2 µL of concentrated bacteriophage MS2 RNA. This will prepare an RNA working solution with a final concentration of Concentration5 ng/µL in Amount100 µL .
Keep the tube on ice at all times to maintain RNA stability.
In the 96-well sample plate, add Amount16 µL of nuclease-free water to a designated well.
Add Amount4 µL of the RNA working solution to the same well, pipetting mix with the Amount16 µL of nuclease-free water.
The remaining Amount96 µL of the RNA working solution can be stored at Temperature-80 °C for two additional uses, after which any leftover solution should be discarded.
cDNA APPROACH
cDNA APPROACH
Transfer Amount20 µL negative control extraction to a new tube or a well of a 96-well PCR plate.
Pipetting
Use prepared samples, positive control, and negative control for cDNA SYNTHESIS AND AMPLIFICATION (starting at step 17).
cDNA APPROACH
cDNA APPROACH
Add Amount10 µL nuclease-free water to the Amount10 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 Concentration50 ng/µL to
Concentration300 ng/µL , typically between Concentration80-150 ng/µL .
Pipetting
Mix
cDNA SYNTHESIS AND AMPLIFICAT
cDNA SYNTHESIS AND AMPLIFICAT
3h 7m 20s
3h 7m 20s
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 Temperature80 °C . Set up thermal cycler programs:Temperature37 °C , Duration00:20:00 and hold at Temperature4 °C ..

7m
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).
ABC
ComponentVolume per reactionMaster mix volume (μL)
10× Turbo DNase Buffer3 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 water5.25 μL(5.25 × N)×1.1=
Total volume10 μL(10 × N)×1.1=

Pipetting
Aliquot Amount10 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 Amount20 µL of samples or controls.
Mix
The final volume per well should be Amount30 µL . Seal the plate with a self-adhesive plate-sealing film.
Incubation
Gently mix the samples then centrifuge the sample plate for Duration00:01:00 .
1m
Incubate the sample for Duration00:20:00 at Temperature37 °C and hold at Temperature4 °C .
20m
Incubation
DNA-free RNA purification (~ 30 mins)
Centrifuge the sample plate for Duration00:01:00 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 Duration00:02:00 ).
3m
Temperature
Resuspend the AMPure XP beads by vortexing for Duration00:00:20 .
20s
Carefully remove the plate seal to avoid cross-contamination of the droplets.
Add Amount30 µL of resuspended AMPure XP beads to the reaction wells in the sample plate and mix by pipetting 5-10 times.
Pipetting
Seal the plate with a new self-adhesive plate-sealing film.
Incubation
Temperature
Incubate the sample plate on a rotator mixer at a speed of >1600 rpm for Duration00:05:00 at TemperatureRoom temperature (If a mixer is unavailable, vortex the plate every Duration00:02:00 ).
7m
Pipetting
Centrifuge the sample plate for Duration00:01:00 .
1m
Mix
Place the plate on a magnet for a 96-well plate. Keep the plate on the magnet forDuration00:03:00 to Duration00:05:00 until the supernatants are clear.
8m
Incubation
While on the magnet, carefully remove the plate seal to avoid cross-contamination of the droplets.
Incubation
Pipette off the supernatants without touching the bead pellet.
Temperature
Keep the plate on the magnet and wash the beads with Amount200 µL of freshly prepared 70% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
Pipetting
Repeat the previous step.
Mix
Seal the plate with a new self-adhesive plate-sealing film.
Incubation
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 Amount13 µL nuclease-free water by pipetting for Duration00:05:00 to Duration00:10:00 .
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 Duration00:10:00 at TemperatureRoom 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 Amount11 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 approachDuration00:10:00
10m
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 Temperature-20 °C . Also, resuspend the lyophilized RLB TSO oligo in RNA storage solution at a molar concentration of
Concentration100 micromolar (µM) as stock solution. Aliquot the RLB TSO oligo stock into 1.5 mL Nuclease-free, low-binding tubes for Amount3 µL per tube. Store the aliquots at Temperature-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
  • Concentration2 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 Amount3 µL of Concentration100 micromolar (µM) RLB RT-9N DNA oligo stock with Amount147 µL of nuclease-free water to make aConcentration2 micromolar (µM) working solution. Mix the diluted oligo thoroughly by vortexing. The unused Concentration2 micromolar (µM) working solution can be kept at Temperature-20 °C for repeated use.
  • Making Concentration2 micromolar (µM) RLB TSO oligo: Retrieve a tube of RLB TSO RNA oligo aliquot (Amount3 µL ) from the Temperature-80 °C freezer. Add Amount147 µL of nuclease-free water directly to the tube to prepare the Concentration2 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 Concentration10 millimolar (mM) dNTP, along with the 5× SuperScript IV RT buffer and Concentration0.1 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 Temperature100 °C . Set up two programs as follows,
  • 1) RNA denature: Temperature65 °C , Duration00:05:00 , then hold at Temperature4 °C .
  • 2) Template switch RT: Temperature50 °C forDuration01:30:00 , Temperature70 °C for Duration00:10:00 , then hold atTemperature4 °C

Prepare a master mix of Concentration10 millimolar (mM) dNTP and Concentration2 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).
ComponentVolume per reactionMaster mix volume (μL)
10 mM dNTP 1 μL(1 × N)×1.2=
2 μM RLB RT 9N 1 μL(1 × N)×1.2=
Total volume2 μL(2 × N)×1.2=
Vortex the master mix gently, then spin down.
Aliquot Amount2 µL master mix into each purified Amount10 µL DNA-free RNA sample in the plate prepared in Step 40. Mix well the Amount12 µL reactions by pipetting.
Seal the plate with a new self-adhesive plate-sealing film.
Heat the plate on the thermocycler to Temperature65 °C for Duration00:05:00 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).
ABC
ComponentVolume per reactionMaster mix volume (μL)
5× RT buffer4 μL(4 × N)×1.1=
RNase inhibitor1 μL(1 × N)×1.1=
2 μM RLB TSO oligo1 μL(1 × N)×1.1=
0.1M DTT1 μL(1 × N)×1.1=
SuperScript IV reverse transcriptase (SS4RT)1 μL(1 × N)×1.1=
Total volume8 μL(8 × N)×1.1=
Vortex the master mix gently, then spin down.
Make sure the plate in Step 49 has been cooled at Temperature4 °C . Place the plate TemperatureOn ice .
Carefully remove the plate seal. Aliquot Amount8 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 Temperature50 °C for Duration01:30:00 followed by Temperature70 °C for Duration00:10:00 and then hold at Temperature4 °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
Concentration100 micromolar (µM) as stock solution. Store the DNA oligo stock at Temperature-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 Concentration10 micromolar (µM) RLB PCR DNA oligo: Thaw the
  • Concentration100 micromolar (µM) oligo stock TemperatureOn ice . In a new RNase-free low-binding 1.5 mL tube, combine Amount10 µL of oligo stock with Amount90 µL of nuclease-free water to make a Concentration10 micromolar (µM) working solution. Mix the diluted oligo thoroughly by vortexing. The unused Concentration10 micromolar (µM) working solution can be kept at Temperature-20 °C for repeated use.
  • Set up thermal cycler: Set lid temperature atTemperature100 °C , and set up the cDNA amplification program following the table below.
StepTemp (༠C)DurationCycle
Initial denaturation953 mins1
Denaturation9815 secs27
Annealing6215 secs
Extension655 mins
Final extension6510 mins1
Storage4Hold1
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).
ComponentVolume per reactionMaster mix volume (μL)
10 μM RLB PCR oligo2 μL(2 × N)×1.1=
Q5 High-Fidelity 2X Master Mix15 μL(15 × N)×1.1=
Nuclease-free water 8 μL(8 × N)×1.1=
Total volume25 μL(25 × N)×1.1=
Vortex the master mix gently, then spin down.
In a new 96-well PCR plate, aliquot Amount25 µL of the PCR reaction master mix into the wells designated for each sample.
Add Amount5 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 Temperature-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 Temperature-20 °C before purification.
cDNA SYNTHESIS: Purification of double-stranded cDNA (~ 30 mins)
cDNA SYNTHESIS: Purification of double-stranded cDNA (~ 30 mins)
19m 30s
19m 30s
Purification of amplified double-stranded cDNA (~ 30 mins):
Note
NOTE: Before starting, prepare fresh 70% ethanol in nuclease-free water sufficient for your samples. (Amount500 µL per sample).

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. (Amount500 µL per sample).
Centrifuge the sample plate for Duration00:02:00 (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 Duration00:03:00 ).

5m
Add Amount30 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.
Pipetting
Mix
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 Duration00:05:00 at TemperatureRoom temperature .(If a mixer is unavailable, vortex the plate every 2 minutes).
5m
Centrifuge the plate for Duration00:02:00 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 tube on the magnet and wash the beads with Amount200 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 Duration00:01:00 and place the plate back on the magnet. Using a pipette, remove any residual ethanol. Allow to dry for ~Duration00:00:30 , 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 Amount13 µL nuclease-free water by slow pipetting to elute the cDNA.
Seal the plate with a new self-adhesive plate-sealing film.
Optional
Incubate on a rotator mixer at speed of>1600 rpm) for Duration00:05:00 at TemperatureRoom temperature (If a mixer is unavailable, vortex the plate every 2 minutes).
5m
Centrifuge the plate for Duration00:01:00 and place the plate back on the magnet until the eluate is clear and colorless.
1m
Remove and retain Amount11 µL of eluate into a new 96-well PCR plate.
Analyze Amount1 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 Temperature-20 °C before sequencing.


SEQUENCING LIBRARY PREPARATION
SEQUENCING LIBRARY PREPARATION
10m
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 TemperatureOn 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) TemperatureOn 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 (Amount1 mL per sample).
  • Prepare fresh 80% ethanol in nuclease-free water (Amount1.5 mL per sample).
  • Program a thermal cycler: Temperature20 °C for Duration00:05:00 and Temperature65 °C forDuration00:05:00 .
  • 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 Amount20 µL nuclease-free water.

10m
SEQUENCING LIBRARY PREPARATION: End-prep (~ 50 minutes)
SEQUENCING LIBRARY PREPARATION: End-prep (~ 50 minutes)
19m 30s
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).
ComponentVolume per reactionMaster mix volume (μL)
Nuclease-free water 4 μL(4 × N)×1.2=
Ultra II End-prep reaction buffer1.75 μL(1.75 × N)×1.2=
Ultra II End-prep enzyme mix1.5 μL(1.5 × N)×1.2=
NEBNext FFPE DNA Repair Buffer 1.75 μL(1.75 × N)×1.2=
NEBNext FFPE DNA Repair Mix1 μL(1 × N)×1.2=
Final total volume10 μL(10 × N)×1.2=
Pipetting
Spin down the plate, and remove the plate seal carefully. Aliquot Amount10 µL of the master mix to each well of a 96-well PCR plate containing Amount20 µL of samples or controls. Mix Amount30 µL End-prep reactions gently by pipetting and spin down.
Mix
Using a thermal cycler, incubate at Temperature20 °C for Duration00:05:00 and Temperature65 °C for Duration00:05:00 .
10m
Incubation
Resuspend the AMPure XP beads by vortexing.
Add Amount50 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).
Pipetting
Mix
Incubate on a Hula mixer (a rotator mixer >1600 rpm) for Duration00:05:00 at TemperatureRoom temperature . (If a mixer is unavailable, vortex the plate every 2 minutes)
5m
Seal the plate with a new self-adhesive plate-sealing film.
Centrifuge the plate forDuration00:02:00 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 Amount200 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.
Spin down and place the tube back on the magnet. Using a pipette, remove any residual ethanol. Allow to dry for ~Duration00:00:30 , but do not dry the pellet to the point of cracking.
30s
Remove the tube from the magnetic rack and resuspend the pellet in Amount12 µL nuclease-free water by pipetting.
Seal the plate with a new self-adhesive plate-sealing film.
Incubate for Duration00:02:00 at TemperatureRoom temperature .
2m
Spin down and place the tube back on the magnet until the eluate is clear and colorless.
Remove the seal and transfer Amount11 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).
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 Temperature-20 °C before barcode ligation.
SEQUENCING LIBRARY PREPARATION: Barcode ligation (~ 25 minutes)
SEQUENCING LIBRARY PREPARATION: Barcode ligation (~ 25 minutes)
20m
20m
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.
AB
Component Volume
End-prepped DNA10 μl
Native Barcode (pick one form Native Barcoding Expansion 1-96)2 μl
Blunt/TA Ligase Master Mix12 μl
Final total volume24 μl
Mix gently by pipetting 10-15 times, seal, and spin down the plate.
Incubate the reaction for Duration00:20:00 at TemperatureRoom temperature .
20m
Spin down the plate and remove the plate seal. Add Amount3 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
SEQUENCING LIBRARY PREPARATION: Library pooling for multiplex sequencing
10m
10m
Resuspend the AMPure XP beads by vortexing.
Use the table below to pool the barcoded samples in a new Amount1.5 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.
ABC
MinION (R10.4.1)PromethION (R10.4.1)
Each barcoded sample (μL) 1212
Number of samples in a pool 2448
Blunt/TA Ligase Master Mix (NEB M0367L, ready-to-use)288576
Total volume of a pool (μL) AMPure XP beads for purification (μL, 0.5x of the pooled library volume)144288
Incubate on a Hula mixer (or a rotator mixer) for Duration00:10:00 at TemperatureRoom temperature .
10m
Spin down the sample and pellet on a magnet. Keep the tube on the magnet for Duration00:05:00 , and using a pipette, discard the supernatant.
5m
Keep the tube on the magnet and wash the beads with Amount700 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 ~Duration00:00:30 , but do not dry the pellet to the point of cracking.
30s
Remove the tube from the magnetic rack and resuspend the pellet in Amount32 µL nuclease-free water. Incubate for Duration00:10:00 at Temperature37 °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
Incubation
Spin down and pellet the beads on a magnet until the eluate is clear and colorless.
Remove and retain Amount31 µL of eluate into a clean Amount1.5 mL low DNA binding tube.
SEQUENCING LIBRARY PREPARATION: Adapter ligation (~ 45 minutes)
SEQUENCING LIBRARY PREPARATION: Adapter ligation (~ 45 minutes)
20m
20m
BEFORE STARTING: Thaw Short Fragment Buffer (SFB), Elution Buffer (EB), and NEBNext Quick Ligation Reaction Buffer (5×) at TemperatureRoom temperature , mix by vortexing, spin down, and place TemperatureOn 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 TemperatureOn ice .
Taking the pooled and barcoded DNA, perform adapter ligation as follows, mixing by flicking the tube between each sequential addition.
AB
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 Duration00:20:00 at TemperatureRoom temperature .
20m
Resuspend the AMPure XP beads by vortexing.
Add Amount50 µL of resuspended AMPure XP beads to the reaction and mix by pipetting.
Pipetting
Mix
Incubate on a Hula mixer (rotator mixer) for Duration00:10:00 at TemperatureRoom temperature .
10m
Place on the magnetic rack, allow beads to pellet and using a pipette, discard the supernatant.
Add Amount125 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.
ABC
MinION (R10.4.1)PromethION (R10.4.1)
EB (μL)1333
Incubate at Temperature37 °C for Duration00:10:00 , agitate the sample for 10s every 2 min.
10m
Incubation
Pellet beads on magnet until the eluate is clear and colorless.
Remove and retain Amount13 µL (MinION) or Amount33 µL (PromethION) of eluate into a clean Amount1.5 mL low DNA binding tube.
Quantify Amount1 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.
ABC
MinION (R10.4.1)PromethION (R10.4.1)
Library volume1232
Expected recovery quantity (ng)100-400200-1000
Expected library molar conc. (fmol)30100
Put the library TemperatureOn ice until ready to load or store the library at Temperature-20 °C for future sequencing.
Priming and loading the SpotON Flow Cell
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 Temperature4 °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
Priming and loading the SpotON Flow Cell: Flow cell priming
5m
5m
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 1 for the positions of the flow cell ports.

Check for a small air bubble under the cover. Draw back a small volume (Amount20-30 µL ) to remove any bubbles:
Set a P1000 pipette to Amount200 µL . Insert the tip into the priming port. Turn the volume adjustment wheel counter-clockwise until the dial shows Amount220-230 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 Amount20-30 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 Amount2.0 mL low DNA binding tube, prepare flow cell priming mix with components as follows, mix by inverting the tube and pipetting.
ABC
Component MinIONPromethION
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 Amount800 µL (MinION) or Amount500 µL (PromethION) of the priming mix into each flow cell via the priming port, avoiding the introduction of air bubbles. Wait for Duration00:05:00 .

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:
ABC
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 Amount200 µL (MinION) or Amount500 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 2d
Priming and loading the SpotON Flow Cell: Data acquisition and basecalling 2d
2d
2d

Note
NOTE: A series of snapshots showing the way to MinKNOW is listed in Appendix 2.

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 Duration48:00:00 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 Amount1 mL pipette to remove Amount1 mL waste solution in the waste channel via waste port 1 (see Appendix 1 under Guidelines & Warnings tab). Remove the flow cells on the device, put it back in the original package, and turn off the device.
Protocol references
REFERENCES

REDI-NET Overview Summary

Double-stranded cDNA synthesis (NEB first and second strand cDNA synthesis protocols):
  • NEBNext Ultra II RNA First Strand synthesis manual E7771
  • NEBNext Ultra II Non-directional RNA Second Strand synthesis manual E6111
  • ezdnase_PI

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