Dec 17, 2024
Version 1
REDI-NET FF-4 FILTH FLY TESTING V.1
- 1REDI-NET Consortium
- Remote Emerging Disease Intelligence - NETwork (REDI-NET)
Protocol Citation: REDI-NET Consortium 2024. REDI-NET FF-4 FILTH FLY TESTING. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvw17o2lmk/v1
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: December 17, 2024
Protocol Integer ID: 107438
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:
- 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.
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
| 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 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.
| 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 # | |
| 200 ng DNA from a sample | Per sample from SOP S-2 (gDNA Preparation) | REDI-NET DNA sample | |
| 20 ul eluents from negative control extraction | From SOP S-2 (gDNA Preparation) | REDI-NET negative control | |
| 100 ng DNA from positive control extraction | From SOP S-2 (gDNA Preparation) | REDI-NET positive control | |
| 160 ng RNA from a sample | Per sample from SOP S-2 (TNA preparation) | REDI-NET RNA sample | |
| 40 ng RNA from positive control extraction | from SOP S-2 (TNA preparation) | REDI-NET negative control | |
| 8 µl total nucleic acid negative control extraction | From SOP S-2 (TNA preparation) | REDI-NET positive control | |
| 10 µl total nucleic acid | Per sample from SOP S-2 (TNA Preparation) | REDI-NET TNA sample | |
| 10 µl total nucleic acid from negative control extraction | From SOP S-2 (TNA Preparation) | REDI-NET negative control | |
| 10 µl total nucleic acid from positive control extraction | from SOP S-2 (TNA Preparation) | REDI-NET positive control | |
| Native Barcoding Kit 96 V14 | (Sequencing Library Preparation) | Oxford Nanopore, SQK-NBD114.96 | |
| ezDNase | (cDNA synthesis) | ThermoFisher, Invitrogen 11766051 | |
| NEBNext Ultra II RNA First Strand Synthesis Module | (cDNA synthesis) | New England Biolabs, E7771L | |
| NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module | (cDNA synthesis) | New England Biolabs, E6111L | |
| Random primer mix (Random hexamer and poly-T mixture) | (cDNA synthesis) | New England Biolabs, S1330 | |
| USB Dithiothreitol (DTT), 0.1M Solution | (cDNA synthesis) | ThermoFisher,707265ML | |
| 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 | |
| Ultra pure Bovine Serum Albumine (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 |
Safety warnings
RISKS AND PERSONAL PROTECTION
Gloves should be worn all the time when handling samples.
Before start
BEFORE START
- Must check the DNA and RNA concentrations of your samples of total nucleic acid (TNA).
- The sequencing approach should be selected based on the target of interest. For viral targets, use cDNA to prepare the sequencing library. For bacterial targets, use gDNA. If both viral and bacterial targets are of interest, use TNA to prepare the sequencing library.
- 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.
- 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.
| 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) | |
| 160 ng, RNA | x | 8-x | 8 | |
| 200 ng, DNA | x | 10-x | 10 |
| A | B | C | D | |
| RNA amount (cDNA approach) | Sample volume | Nuclease-free water (μL) | Total volume (μL) | |
| 160 ng, RNA | x | 8-x | 8 |
5. Use the following table to prepare positive controls for the corresponding
sequencing approach.
| A | B | C | D | |
| DNA amount (gDNA approach) | Sample volume | Nuclease-free water (μL) | Total volume (μL) | |
| 50 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 | 8-x | 8 | |
| 50 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 | 8-x | 8 |
gDNA APPROACH
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.
Prepare 50 ng gDNA from positive control extraction in 20 µL 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 2.5 ng/µl , directly transfer 20 µL positive control eluent to a tube or a well.
Transfer 20 µL negative control extraction to a new tube or a well of 96-well PCR plate.
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 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 50 ng of gDNA from a positive control extraction 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 5 ng/µl , directly transfer 10 µL of the positive control eluent to the tube or well.
Transfer 10 µL of the negative control extraction to a new tube or a well of a 96-well PCR plate.
Prepare cDNA from RNA following the quantities outlined in the tables listed in the "Before Start" section, and the procedure outlined in the cDNA SYNTHESIS section (starting at step 17).
Add the corresponding 10 µL of purified double-stranded cDNA from the sample, positive control, and negative control to the 10 µL DNA as prepared in steps 6-8.
Subject the 20 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 160 ng of DNA in 8 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 20 ng/µl , directly transfer 8 µL of the sample to the tube or well.
Prepare 40 ng RNA from positive control extraction and adjust the volume to final 8 µL with 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 5 ng/µl , directly transfer 8 µL of the positive control eluent to the tube or well.
Transfer 8 µL negative control extraction to a new tube or a well of 96-well PCR plate.
Use prepared 8 µL samples, positive control, and negative control for cDNA SYNTHESIS (starting at step 17).
cDNA APPROACH
cDNA APPROACH
Add 10 µL nuclease-free water to the 10 µL purified double-stranded cDNA for SEQUENCING LIBRARY PREPARATION.
cDNA SYNTHESIS
cDNA SYNTHESIS
5m
5m
Remove contaminated DNA (~ 15 mins):
Thaw total nucleic acid and 10x ezDNase Buffer on ice at room temperature. Vortex 10x ezDNase Buffer briefly, spin done by centrifugation for 5 seconds, and place on ice. ezDNase is not frozen and should be placed on ice before use. Set up thermal cycler programs: 37 °C , 00:02:00 and 55 °C , 00:05:00 .
7m
Mix the following components in an RNase-free tube or plate. For processing multiple samples, make master mix for 10X ezDNase buffer and ezDNase with 10% overage. Aliquot the master mix into the wells of a 96-well plate, then add TNAs.
| A | B | |
| Component | Volume | |
| 10x ezDNase Buffer | 1 μl | |
| ezDNase | 1 μl | |
| RNA from step 12 | 8 μl | |
| Total volume | 10 μl |
Gently mix the samples then centrifuge the tube (Include a reaction for extraction positive control and negative control of each batch nucleic acid extraction).
Incubate the sample for 00:02:00 at 37 °C .
2m
Add 1 µL 100 millimolar (mM) DTT into the reaction tube.
Incubate the sample at 55 °C for 00:05:00 to inactivate the enzyme.
5m
Chill the tube on ice to bring the sample to room temperature, then spin down and place the tube on ice.
BEFORE START: Thaw 60 micromolar (µM) stock Random Primer Mix (NEB, S1330S) at Room temperature . DO NOT USE the Random Primer provided by the NEBNext First Strand Synthesis Module. Thaw Random Primer Mix solution, NEBNext First Strand Reaction Buffer, NEBNext Second Strand Reaction Buffer at Room temperature then place On ice . Vortex the vials briefly, spin done by centrifugation for 00:00:05 , and place On ice . First and Second Strand Enzyme Mix are not frozen, should be briefly centrifuged and placed On ice before use.
5s
Add the following reagents into a sterile, RNase-free 0.2 mL PCR tube on ice. For processing multiple samples, make a master mix for the 60 micromolar (µM) Random Primer Mix and nuclease free water with 10% overage. Aliquot the master mix into the wells of a 96-well plate, then add TNAs.
| A | B | |
| Component | Volume | |
| ezDNase treated RNA | 10 μl | |
| 60 μM Random Primer | 1 μl | |
| Nuclease free water | 3 μl | |
| Total volume | 14 μl |
Mix gently, spin down and incubate at 65 °C for 00:05:00 . Chill On ice , spin down again and place On ice .
5m
Add the following components in the indicated order, if multiple reactions will be processed at the same time, make a master mix with a 10% overage:
| A | B | |
| Component | Volume | |
| NEBNext First Strand Synthesis Reaction Buffer | 4 μl | |
| NEBNext First Strand Synthesis Enzyme Mix | 2 μl | |
| Total volume | 20 μl |
Mix gently and spin down.
Incubate the tube for 00:10:00 at 25 °C followed by 00:15:00 at 42 °C .
25m
Terminate the reaction by heating at 70 °C for 00:15:00 .
15m
Place the tube On ice or pre-chilled freezer block.
Pipette the following components directly into the first strand reaction tube (with 20 µL mixture) On ice in the indicated order, if multiple reactions will be processed at the same time, make a master mix with a 10% overage:
| A | B | |
| Component | Volume | |
| 5x NEBNext Second Strand Synthesis Reaction Buffer | 5 μl | |
| NEBNext Second Strand Synthesis Enzyme Mix | 2.5 μl | |
| Nuclease-free water | 22.5 μl | |
| Final total volume | 50 μl |
Mix gently and centrifuge briefly.
Incubate at 16 °C for 01:00:00 (heated lid set at ≤ 40 °C ).
1h
Proceed with cDNA purification or store the reaction mixture at -20 °C before the subsequent cDNA purification.
cDNA SYNTHESIS: Purification of double-stranded cDNA (~ 15 mins)
cDNA SYNTHESIS: Purification of double-stranded cDNA (~ 15 mins)
5m
5m
Note
NOTE: Before starting, prepare fresh 70% ethanol in nuclease-free water sufficient for your samples. (500 µL per sample).
Resuspend the AMPure XP beads by vortexing.
Transfer the sample (50 µL ) to a clean 1.5 mL low DNA binding tube.
Add 40 µL of resuspended AMPure XP beads to the reaction and mix by flicking the tube.
Incubate on a Hula mixer (or a rotator mixer >1600 rpm) for 00:05:00 at Room temperature .
5m
Spin down the sample and pellet on the magnet. Keep the tube on the magnet, and using a pipette, discard the supernatant.
Keep the tube 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.
Spin down and place the tube back on the magnet. Pipette off 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 13 µL nuclease-free water.
Incubate on a Hula mixer (rotator mixer) for 00:10:00 at Room temperature .
10m
Spin down and pellet beads on magnet until the eluate is clear and colorless.
Remove and retain 11 µL of eluate into a clean 1.5 mL low DNA binding tube.
Optional: : If using the cDNA sequencing approach for viral target identification, analyze 1 µL of the purified double-stranded cDNA for quantity using Qubit fluorometer and Qubit 1X dsDNA HS Assay Kit.
Use the purified 10 µL cDNA for the cDNA or TNA sequencing approach, see TNA APPROACH or cDNA APPROACH.
Note
STOP POINT: The synthesized double-stranded cDNA can be stored at -20 °C before sequencing.
SEQUENCING LIBRARY PREPARATION
SEQUENCING LIBRARY PREPARATION
10m
10m
Before starting, prepare fresh 70% ethanol in nuclease-free water sufficient for your samples (1 mL per sample). Program the thermal cycler or use a heat block for 96 well plate: 20 °C for 00:05:00 and 65 °C for 00:05:00 . Thaw Ultra II End-prep reaction buffer, NEBNext FFPE DNA Repair Buffer, Barcode Plate(from SQK-NBD114.96 Kit),and Blunt/TA Ligase Master Mix On ice . After fully thaw, mix by vortex, spin down briefly, and place On ice . Check that there is no precipitate present (the Blunt/TA Master Mix can sometimes form a precipitate). Spin down Ultra II End-prep enzyme mix and place On ice .
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)
SEQUENCING LIBRARY PREPARATION: End-prep (~ 50 minutes)
10m
10m
Mix the following reagents for one sample. When working with 24 or 48 samples, prepare a master mix by multiplying the ingredients (except gDNA/cDNA/TNA) with a 10% overage. Aliquot 10 µL of the master mix for each sample in a 96 well plate, then add the prepared samples:
| A | B | |
| Component | Volume | |
| DNA/TNA sample | 20 μl | |
| Nuclease-free water | 4 μl | |
| Ultra II End-prep reaction buffer | 1.75 μl | |
| Ultra II End-prep enzyme mix | 1.5 μl | |
| NEBNext FFPE DNA Repair Buffer | 1.75 μl | |
| NEBNext FFPE DNA Repair Mix | 1 μl | |
| Final total volume | 30 μl |
Mix 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 .
5m
Spin down the sample and pellet on a magnet (DynaMag-2 for 1.5 mL tube and DynaMag-96 for PCR plate). Keep the tube on the magnet, and using a pipette, discard the supernatant.
Keep the tube 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.
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 11 µL nuclease-free water. Incubate for 00:02:00 at Room temperature .
2m
Pellet the beads on a magnet until the eluate is clear and colorless.
Remove and retain 10 µL of eluate into a clean 1.5 mL low DNA binding tube.
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.
| 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 flicking the tube and spin down.
Incubate the reaction for 00:20:00 at Room temperature .
20m
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.
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 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 31 µL nuclease-free water. Incubate for 00:10:00 at 37 °C temperature.
10m
Spin down and pellet the beads on a magnet until the eluate is clear and colorless.
Remove and retain 30 µL of eluate into a clean 1.5ml 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 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, mixing 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 00:20:00 at Room temperature .
20m
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 at 37 °C for 00:10:00 , agitate the sample for 10s every 2 min.
10m
Pellet beads on magnet until the eluate is clear and colorless.
Remove and retain 13 µL 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 |
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
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
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 Room temperature . Mix SB by tapping or pipetting (DO NOT Vortex) and vortex the other tubes. Spin down tubes at Room 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 (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 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 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
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 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
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