Feb 02, 2024
SW-4 SWAB TESTING
- REDI-NET Consortium1
- 1REDI-NET Consortium
Protocol Citation: REDI-NET Consortium 2024. SW-4 SWAB TESTING. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj38mnlk5/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: August 09, 2023
Last Modified: March 15, 2024
Protocol Integer ID: 86263
Keywords: gDNA PREPARATION, TNA PREPARATION, cDNA SYNTHESIS, Purification of double-stranded cDNA, SEQUENCING LIBRARY PREPARATION
Funders Acknowledgement:
USAMRAA
Grant ID: W81XWH-21-C-0001
USAMRAA
Grant ID: W81XWH-22-C-0093
USAMRAA
Grant ID: HT9425-23-C-0059
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 and HT9425-23-C-0059. 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 protocol details standard operating procedure for swab testing.
Guidelines
OBJECTIVE
To outline the procedures for properly using the Oxford Nanopore Sequencing platforms (GridION or MinION Mk1C) to sequence gDNA and TNA extracted from collected swab 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
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
APPENDICES
APPENDIX 1. FLOW CELL
APPENDIX 2. 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 3. 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 | Oxford Nanopore Technologies, GRD-CapEx or Oxford Nanopore Technologies, M1CCapEx | |
| 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 | |
| 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 B-2 (gDNA Preparation) | REDI-NET DNA sample | |
| 20 ul eluents from negative control extraction | From SOP B-2 (gDNA Preparation) | REDI-NET negative control | |
| 100 ng DNA from positive control extraction | From SOP B-2 (gDNA Preparation) | REDI-NET positive control | |
| 160 ng RNA from a sample | Per sample from SOP B-2 (TNA preparation) | REDI-NET RNA sample | |
| 40 ng RNA from positive control extraction | from SOP B-2 (TNA preparation) | REDI-NET negative control | |
| 8 µl total nucleic acid negative control extraction | From SOP B-2 (TNA preparation) | REDI-NET positive control | |
| 10 µl total nucleic acid | Per sample from SOP B-2 (TNA Preparation) | REDI-NET TNA sample | |
| 10 µl total nucleic acid from negative control extraction | From SOP B-2 (TNA Preparation) | REDI-NET negative control | |
| 10 µl total nucleic acid from positive control extraction | from SOP B-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 | |
| 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 | |
| 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
ezDNase™ EnzymeThermo FisherCatalog #11766051
NEBNext Ultra II RNA First Strand Synthesis Module - 96 rxnsNew England BiolabsCatalog #E7771L
NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module - 100 rxnsNew England BiolabsCatalog #E6111L
Random primer mixNew England BiolabsCatalog #S1330S
USB Dithiothreitol (DTT) 0.1M SolutionThermo Fisher ScientificCatalog #707265ML
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
96 well LoBind PCR plates Semi-skirtedEppendorfCatalog #0030129504
NEBNext Microbiome DNA Enrichment Kit - 6 rxnsNew England BiolabsCatalog #E2612S the
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.
Before start
BEFORE START
- Check the DNA and RNA concentrations in each sample of total nucleic acid (TNA) extraction.
- If the concentrations are detectable, choose the sequencing approach following the table below.
- If DNA or RNA concentration is not detectable, prepare the library for the detectable one.
- Use sections gDNA PREPARATION and TNA PREPARATION for gDNA and TNA preparation, respectively, then subject the prepared gDNA and TNA to Section SEQUENCING LIBRARY PREPARATION.
| A | B | C | D | E | |
| DNA concentration (ng/ul) | |||||
| < 1 ng/ul | 1-10 ng/ul | > 10 ng/ul | |||
| RNA concentration (ng/ul) | < 4 ng/ul | TNA | DNA | DNA | |
| 4-20 ng/ul | TNA | TNA | TNA | ||
| >20 ng/ul | TNA | TNA | TNA | ||
gDNA PREPARATION
gDNA PREPARATION
When the RNA concentration of the sample is lower than the detectable range of the Qubit High Sensitivity Assay (<0.01 ng/µl ), the sample is subjected to gDNA sequencing. The cDNA synthesis can be skipped.
When the DNA concentration >10 ng/ul , calculate the required volume of 200 ng DNA, then transfer the volume to a new 200μl 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 .
Prepare 100 ng gDNA from positive control extraction in 20 µL nuclease-free water in a new 200μl PCR tube or a well of a 96-well PCR plate.
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 PREPARATION
TNA PREPARATION
To prepare TNA for sequencing both cDNA and gDNA, cDNA needs to be prepared separately and then mixed with TNA from the original sample.
Prepare cDNA following section cDNA SYNTHESIS (positive control and negative control included) until step 40.
Transfer 10 µL double-stranded cDNA of section cDNA SYNTHESIS step 40 to a new 200 μl PCR tube or a well of a 96-well PCR plate. Add 10 µL of TNA from the original sample to make the final volume 20 µL .
Subject the 20 µL double-stranded cDNA/TNA mixture to section SEQUENCING LIBRARY PREPARATION.
Note
NOTE: Twenty-four samples must be pooled in one sequencing run to make the most out of a sequencing flow cell. For collecting 24 samples, the samples from gDNA and TNA preparations can be placed in the same 96-well plate for End-prep and Barcode Ligation before pooling, then pooled for the subsequent steps of library preparation.
cDNA SYNTHESIS: DNase treatment
cDNA SYNTHESIS: DNase treatment
If RNA concentration > 20 ng/µl , calculate the required volume of 160 ng RNA,then transfer the volume to a new 200μl PCR tube or a well of a 96-well PCR plate. Adjust the volume with nuclease-free water to a final volume of 8 µL . If the concentration of the RNA < 20 ng/µl , directly transfer 8 µL of RNA to a new 200μl PCR tube or a well of a 96-well PCR plate. Keep the tube or plate On ice .
Prepare 40 ng RNA from positive control extraction and adjust the volume to final 8 µL with nuclease-free water in a new 200μl PCR tube or a well of a 96-well PCR plate.
Transfer 8 µL negative control extraction to a new tube or a well of a 96-well PCR plate.
Remove contaminated DNA (~ 15 mins): Thaw total nucleic acid, 10x ezDNase Buffer, and DTT on the ice at Room temperature . Vortex 10x ezDNase Buffer and DTT briefly, spin down by centrifugation for 00:00:05 , 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 5s
Mix the following components in an RNase-free tube or plate. For processing multiple samples, make a master mix for 10× 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 | |
| 10× ezDNase Buffer | 1 μl | |
| ezDNase | 1 μl | |
| RNA from step 6 | 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 100mM 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 .
cDNA SYNTHESIS: First strand cDNA Synthesis (~ 1hr)
cDNA SYNTHESIS: First strand cDNA Synthesis (~ 1hr)
BEFORE START: Thaw 60 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 the ezDNase-treated RNA from step 13.6. For processing multiple samples, make a master mix for the 60 μM Random Primer Mix and nuclease-free water with 10% overage.
| 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.
Continue immediately with the second strand synthesis reaction as described below.
cDNA SYNTHESIS: Second strand cDNA Synthesis (~ 1hr)
cDNA SYNTHESIS: Second strand cDNA Synthesis (~ 1hr)
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 (the double-stranded cDNA is ready to be shipped to Gold Labs if necessary).
cDNA SYNTHESIS: Purification of double-stranded cDNA (~ 15 mins)
cDNA SYNTHESIS: Purification of double-stranded cDNA (~ 15 mins)
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 1.5ml 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 (rotator mixer) 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. 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 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 1.5ml low DNA binding tube.
Optional: Analyze 1 µL of the purified double-stranded cDNA for quantity using Qubit fluorometer and Qubit 1X dsDNA HS Assay Kit.
Subject 10 µL purified double-stranded cDNA for section SEQUENCING LIBRARY PREPARATION.
Note
STOP POINT: The synthesized double-stranded cDNA can be stored at -20 °C before sequencing.
SEQUENCING LIBRARY PREPARATION
SEQUENCING LIBRARY PREPARATION
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-NBD119.96 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 .
10m
SEQUENCING LIBRARY PREPARATION: End-prep (~ 50 minutes)
SEQUENCING LIBRARY PREPARATION: End-prep (~ 50 minutes)
Mix the following reagents in a 0.2ml PCR tube. To process 24 samples, prepare a master mix by multiplying gradients except for cDNA by 24 with a 10% overage. Aliquot the master mix into a 96-well plate, then add cDNA or TNA (see Appendix 2 for master mix preparation):
| 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 (rotator mixer) for 00:05:00 at Room temperature .
5m
Spin down the sample and pellet on a magnet (DynaMag-2 for 1.5ml 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 12 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 11 µL of eluate into a clean 1.5ml low DNA binding tube.
SEQUENCING LIBRARY PREPARATION: Barcode ligation (~ 25 minutes)
SEQUENCING LIBRARY PREPARATION: Barcode ligation (~ 25 minutes)
Add the reagents in the order given below, mixing by flicking the tube between each sequential addition:
Note
NOTE: When working on 24 End-prepped gDNA/TNA, set up the reactions in a low DNA binding 96-well plate. The Native barcodes can be transferred by an 8-channel pipette directly punching through the sealing foil with tips of the barcode plate. Please reseal the used wells with trimmed adhesive foil. Each well provides sufficient volume for two barcoding ligations.
| 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
Pool every 24 barcoded samples 12 µL from each sample, (total 288 µL /pool) in a new 1.5ml low DNA binding tube.
Resuspend the AMPure XP beads by vortexing.
Add 518 µL (1.8x volume of the pooled library) of resuspended AMPure XP beads to the pooled library and mix by pipetting.
Incubate on a Hula mixer (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 35 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 35 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)
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 20 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 13 µL of Elution Buffer (EB).
Incubate on at 37 °C for 00:10:00 at Room temperature , 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 1.5ml low DNA binding tube.
Quantify 1 µL of eluted sample using a Qubit fluorometer and Qubit 1X dsDNA HS Assay Kit (recovery aim ~ 430 ng in total).
Make up the library to 12 µL at 10-20 fmol.
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.
Turn on GridION (or MinION Mk1C) device. Make sure all the connections for the display, mouse, keyboard, and internet are ready.
Depending on the number of pooled samples, get one to four new flow cells from the fridge and check the expiration date.
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 GridION (or MinION Mk1C) 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.
Select the flow cells to assign the flow cell type FLO-MIN114 from the dropdown menu.
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 pores should not be used for the sequencing. If the flow cell is not expired, contact Oxford Nanopore Company for customer service.
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
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.
Slide open the GridION lid (or MinION Mk1C) and insert flow cell with minimum 800 pores.
Slide the priming port cover clockwise to open the priming port.
Note
NOTE: Please see Appendix 1 for the positions of the flow cell ports.
After opening the priming port, 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 wheel 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.
Prepare flow cell priming mix with components as follows, mix by inverting the tube and pipetting.
| A | B | |
| Component | Volume | |
| Bovine Serum Albumin (BSA) (50 mg/ml) | 5 μl | |
| Flow Cell Tether (FCT) | 30 μl | |
| Flow Cell Flush (FCF) | 1170 μl | |
| Final total volume | 1205 μl |
Load 800 µL 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 | |
| Component | Volume | |
| Sequencing Buffer (SB) | 37.5 μl | |
| Library Beads (LIB) | 25.5 μl | |
| DNA library | 12 μl | |
| Final total volume | 75 μl |
Complete the flow cell priming.
Gently lift the SpotON sample port cover to make the SpotON sample port accessible.
Load 200 µL of the priming mix into the flow cell via the priming port (not the SpotON sample port), avoiding the introduction of air bubbles.
Loading samples.
Mix the prepared library gently by pipetting up and down just prior to loading.
Add 75 µL of sample to the flow cell via the SpotON sample port in a dropwise fashion. Ensure each drop flows into the port before adding the next drop.
Gently replace the SpotON sample port cover, making sure the bung enters the SpotON port, close the priming port and replace the GridION lid.
Priming and loading the SpotON Flow Cell: Data acquisition and basecalling
Priming and loading the SpotON Flow Cell: Data acquisition and basecalling
2d
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 using the scheme, [YYYY_MM_DD_Approach(gDNA or TNA)_Sample type (soil, water,… etc.)]
Type in Sample ID (same as experiment name)
Choose flow cell FLO-MIN114 from the drop-down menu.
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. Let adaptive sampling be 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). Remove the flow cells on the device, put it back in the original package, and turn off the device.
Protocol references