Mar 05, 2025
Version 2
Soil Metagenome PacBio V2 V.2
This protocol is a draft, published without a DOI.
- Robert S James1,2,
- Gaetan Benoit3,
- sebastien raguideau2,
- Georgina Alabone2,
- Christopher Quince2,1
- 1Quadram Institute Bioscience;
- 2Earlham Institute;
- 3Pasteur Institute
- Quince_Group
Protocol Citation: Robert S James, Gaetan Benoit, sebastien raguideau, Georgina Alabone, Christopher Quince 2025. Soil Metagenome PacBio V2. protocols.io https://protocols.io/view/soil-metagenome-pacbio-v2-d43g8yjwVersion created by Robert S James
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: August 15, 2024
Last Modified: March 05, 2025
Protocol Integer ID: 123720
Keywords: Metagenomics, Soil, Long-reads, PacBio, Revio
Disclaimer
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Abstract
This protocol describes the sample collection to sequence acquisition workflow for PacBio Revio long-read sequencing of a complex soil sample using SMRT Bell library prep kit 2.0 and Revio SMRT cells.
Guidelines
- Fully equilibriate Ampure XP SPRI beads to Room temperature before use.
- Fully equilibriate Qubit solution to Room temperature before use.
- Fully equilibriate Tape station screen tape and reagents to Room temperature before use.
- Over drying DNA bound to Ampure XP SPRI beads can reduce Sample recovery.
- Additional time must be given for the Ampure XP beads to clear from the adapter ligation reaction due to the high viscosity of the solution. Failure to provide adequate time for the supernatant to clear can result in Sample loss.
Materials
Equipment
- Bench top centrifuge
- Thermal cycler
- Qubit 4 FluorometerThermo Fisher ScientificCatalog #Q33238
- Capillary electrophoresis instrument (e.g. Agilent Tapestation 4200)Contributed by users
- Rotational mixer
- Heat block
- Magnetic rack
- Fridge 4 °C
- Freezer -80 °C
- Ice bucket
- Pipette set (P10, P20, P200, P1000)
- Soil corer
- Soil Sieve
- Soil collection plate
- Plate sealer
- Top pan balance
- Weigh boat
- Spatula
- Measuring cylinder 100 mL
- Conical flask 250 mL
Reagents
- Zymo DNA/RNA ShieldFisher ScientificCatalog #50-125-1706
- FastDNA™ SPIN Kit for SoilMPBioCatalog #116560200-CF
- Monarch RNase ANew England BiolabsCatalog #T3018L
- Ethanol (100%, Molecular Biology Grade)Fisher ScientificCatalog #BP2818500
- Molecular Biology Grade WaterFisher ScientificCatalog #10154604
- Agencourt AMPure XPBeckman CoulterCatalog #A63880
- Qubit 1X dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q33230
- Genomic DNA ReagentsAgilent TechnologiesCatalog #5067-5366
- Genomic DNA ScreenTapeAgilent TechnologiesCatalog #5067-5365
- PacBio SPK 2.0
- Barcoding overhang kit 8A
Consumables
- Eppendorf lo-bind Falcon tubes 5 mL Catalog no. 0030122348
- Eppendorf lo-bind microfuge tubes 1.5 mL Catalog no. 0030108051
- Qubit™ Assay TubesInvitrogen - Thermo FisherCatalog #Q32856
- Thin-walled PCR tubes 0.2 mL Catalog no: AB-2000
- Cryovials 2 mL Cataloge no. 41121704
- Qubit tubes 0.2 mL Cataloge no. Q32856
- P1000 Wide bore pipette tips
- P1000 pipette tips
- P200 Pipette tips
- P20 pipette tips
- P10 pipette tips
- Crushed ice
Safety warnings
Safety information
- Binding Matrix contains components that, when in contact with human tissue, may cause irritation. Wear personal protective equipment to prevent contact with the skin or mucous membranes (gloves, lab coat, and eye protection).
- EtOH (100%) is a highly flammable liquid and vapour. It can cause serious eye irritation. Keep away from heat, hot surfaces, sparks, open flames and other sources of ignition.
Before start
- Dilute the concentrated SEWS-M solution with 100 mL of 100 % (v/v) EtOH before use.
- Prepare 10 mL of fresh 80 % (v/v) EtOH.
1. Soil sample collection and storage
1. Soil sample collection and storage
Collect approximately 15 g of soil using a sterile soil corer or similar device and transfer to a sterile soil sieve and collection plate.
Homogenise the soil sample by passing it through the soil sieve and collecting the output on the collection plate below the sieve. The use of a sterile plate sealer can be used to facilitate sieve homogenisation.
Use a top pan balance, spatular and weigh boat to weigh 10-50 g of homogenised soil Sample and transfer to a 250 mL conical flask. Promptly suspend the soil in 100 mL of Zymo DNA/RNA shield for a final concentration of 100-500 mg/mL .
Incubate for at least 04:00:00 at room temperature or 4 °C Overnight with occasional mixing.
16h
Gently mix the bulk sample by hand to form a homogenous solution then aspirate 1000 µL of the total sample using a P1000 pipette and wide bore tip. Transfer the Sample into a new and sterile 2 mL cryovial and close the lid securely.
Repeat step 5 until the total volume of sample has exhausted or the desired number of aliquots have been achieved.
Snap freeze and store Sample at -80 °C for future use.
2. DNA extraction and sample cleanup
2. DNA extraction and sample cleanup
1h 38m 15s
1h 38m 15s
Defrost the Sample for 00:15:00 On ice prior to commencing DNA extraction.
15m
Transfer 1000 µL of the Sample to a new and clean 1.5 ml lo-bind Eppendorf tube using a P1000 pipette and wide bore pipette tip.
Centrifuge at 5000 x g, 00:04:00 to pellet the Sample .
4m
Aspirate and discard the supernatant without disturbing the pellet.
Resuspend the pellet in 978 µL of sodium phosphate buffer, then transfer the Sample to a new and clean lyzing matrix E tube.
Add 122 µL of MT buffer to the Sample , mix by inversion and incubate On ice for 00:05:00 .
5m
Place the matrix tube in a MPBio FastPrep instrument (or similar) and homogenise for 00:00:10 at 5.0 ms then return the Sample to ice.
10s
Incubate the Sample On ice for 00:05:00 .
5m
Repeat steps 14 and 15 then continue to step 17.
Add 250 µL of protein precipitation solution (PPS) to a new and clean 1.5 mL lo-bind Eppendorf tube and chill On ice .
Remove the lysing matrix-E tube from the ice and centrifuge at 14000 x g, 00:04:00 to pellet the debris.
4m
Decant the supernatant from the lysing matrix E tube into the pre chilled PPS without disturbing the pellet, then mix the Sample by inversion 10 times and place On ice .
Incubate the Sample On ice for 00:10:00 to facilitate protein precipitation.
10m
Remove the Sample from the ice and centrifuge at 14000 x g, 00:02:00 to pellet the protein precipitate.
2m
Homogenise the binding matrix immediately before use. Add 1000 µL of resuspended binding matrix to a clean 5 mL lo-bind tube.
Safety information
- Binding Matrix contains components that, when in contact with human tissue, may cause irritation. Wear personal protective equipment to prevent contact with the skin or mucous membranes (gloves, lab coat, and eye protection).
Gently decant the Sample directly into the binding matrix without disturbing the protein pellet, secure the lid and mix the Sample by inversion until a homogeneous solution has formed.
Incubate the Sample on a rotational mixer at 30 rpm, Room temperature , 00:10:00 .
10m
Add 1000 µL of DES to a new and clean 1.5 mL lo-bind tube and place the tube into an active heat block set to 56 °C .
Remove the Sample from the rotational mixer and transfer 750 µL of sample to an MPbio spin filter using a wide bore P 1000 pipette tip. Return the Sample to the rotational mixer.
Centrifuge the spin filter at 14000 x g, 00:02:00 and discard flow through.
2m
Repeat steps 26 and 27 until all the binding matrix has been processed through the spin filter.
Add 500 µL of prepared SEWS-M buffer to the Sample bound binding matrix, close the lid securely and suspend the beads in the SEWS-M solution by flicking the tube.
Safety information
EtOH (100%) is a highly flammable liquid and vapour. It can cause serious eye irritation. Keep away from heat, hot surfaces, sparks, open flames and other sources of ignition.
Centrifuge the Sample at 14000 x g, 00:02:00 then discard the flow through.
2m
Repeat steps 29 and 30 then continue to step 32.
Centrifuge the Sample at 14000 x g, 00:02:00 to collect excess EtOH.
2m
Remove the spin filter from the catch tube and place it into a new and clean 1.5 mL lo-bind Eppendorf tube.
Allow the binding matrix to air dry for 00:02:00 .
2m
Add 100 µL of 56 °C DES elution buffer to the binding matrix and agitate the spin filter until a slurry has formed.
Incubate the Sample at 56 °C for 00:10:00 with intermittent agitation.
10m
Centrifuge the Sample at 14000 x g, 00:02:00 . Discard the spin filter and retain the Sample eluate.
2m
Allow the Sample to equilibrate to Room temperature and add 1 µL RNase A at 20 mg/mL . Mix tube by flicking and then incubate at Room temperature for 00:02:00 .
2m
Add 60 µL of Room temperature Ampure XP beads to the Sample and mix by flicking the tube until a homogeneous solution has formed.
Incubate the Sample on a rotational mixer for 00:10:00 at Room temperature .
10m
Briefly spin down the Sample and place on a magnetic rack.
Once the Sample has cleared, aspirate and discard the supernatant without disturbing the beads.
Gently add 200 µL of 80 % (v/v) EtOH across the beads and incubate on the magnetic rack for at least 00:00:30 .
30s
Aspirate and discard the supernatant without disturbing the beads.
Repeat steps 43 and 44 then continue to step 46.
Remove the Sample from the magnetic rack and briefly centrifuge at < 1000 x g to collect any residual EtOH then return the Sample to the magnetic rack.
5s
Promptly remove the excess EtOH with a P10 pipette and tip without disturbing the beads.
Allow the beads to air dry for 00:00:30 or until the beads are satin in appearance. Do not over dry the beads, avoid excess EtOH carry over.
30s
Remove the Sample from the magnetic rack and add 46 µL of molecular grade water to the beads.
Resuspend the beads in the water by flicking the tube until a homogenous solution has formed.
Incubate the Sample at 37 °C for 00:10:00 then return the Sample to the magnetic rack.
10m
Once the solution has cleared, Quantify 1 µL of Sample using a Qubit 4 fluorometer or similar device.
PacBio SPK 2.0 library preparation
PacBio SPK 2.0 library preparation
5h 6m
5h 6m
DNA blunting
Prepare the following reaction on ice. Scale the reaction volume by sample number.
| A | B | |
| Reagent | Volume (µl) | |
| Enzyme Dilution Buffer | 4.0 | |
| DNA Prep Additive | 1.0 |
Prepare the following reaction in a 0.2 ml thin walled PCR tube for each sample and return to ice. Use DNA Prep Additive from step 53.
| A | B | |
| Reagent | Volume (µl) | |
| DNA | 45.0 | |
| DNA Prep Buffer | 7.0 | |
| NAD | 1.0 | |
| DNA Prep Additive | 1.0 | |
| DNA Prep Enzyme | 1.0 | |
| Total | 55 |
Mix the sample by flicking and spin down to collect.
Incubate the Sample in a thermal cycler at 37 °C for 00:15:00 then place the sample On ice .
15m
DNA damage repair (FFPE).
Add 2 µL of DNA damage repair mix v2 to each Sample and mix by flicking then spin down to collect.
Incubate at 37 °C for 00:30:00 then place the Sample On ice .
30m
End-repair and A-tailing.
Add 3 µL of End Prep Mix to each Sample mix by flicking and spin down to collect.
Incubate the Sample at 20 °C for 00:30:00 , then at 65 °C for 00:30:00 then place the sample On ice
1h
SMRT bell adapter ligation
Defrost one barcoded overhang SMRT bell adapter per sample, mix by flicking, spin down and place On ice
Add 5 µL of a single barcoded overhang SMRT bell adapter to each Sample , mix by flicking, spin down and place On ice .
Add 30 µL of Ligation mix, 1 µL of Ligation aditive and 1 µL of Ligation Enhancer to each sample, mix by flicking, spin down and return to ice.
Incubate the sample at 20 °C for 01:00:00 then at 4 °C for up to 16 h.
1h
Incubate the Sample at 65 °C for 00:10:00 then place On ice .
10m
Nuclease treatment
Prepare the following reaction and place on ice. Scale the reaction volume by sample number.
| A | B | |
| Reagent | Volume (µl) | |
| Enzyme A | 4.0 | |
| Enzyme B | 1.0 | |
| Enzyme C | 1.0 | |
| Enzyme D | 2.0 |
Add 8 µL of Enzyme mix to eacg Sample and mix by flicking then spin down to collect.
Incubate at 37 °C for 01:00:00 then place the sample On ice .
1h
Spri cleanup.
Add 47.25 µL (0.45x) of SPRI beads at Room temperature and mix by flicking.
Incubate on a rotational mixer for 00:10:00 .
10m
Briefly spin to collect the Sample .
Place the sample on a magnetic rack and allow the solution to fully clear.
Aspirate and discard the supernatant without disturbing the beads.
Add 200 µL of 80 % (v/v) EtOH across the beads and allow to sit for 00:00:30 .
30s
Aspirate and discard the EtOH.
Repeat steps 74 and 75, then continue.
Spin down to collect the tube contents and return to the magentic rack.
Promptly remove residual EtOH with a pippette.
Add 31 µL of PB 1X Elution buffer to the beads and mix by flicking.
Incubate the Sample at37 °C for 00:30:00 with occasional mixing.
30m
Place sample on a magnetic rack and allow the sample to clear.
Aspirate 31 µL of Sample and transfer to a new 1.5 ml lo-bind tube.
Add 1 µL of Sample to 9 µL of water and mix by flicking.
Quantify 2 µL of diluted sample using a Qubit fluorometer and HS assay kit. Quantify 1 µL of diluted library on a tape station or similar fragment analyser.
Dilute SPRI bead clean up
Add 350 µL of SPRI beads to 650 µL of 1 x PB elution buffer and mix by flicking.
Dilute the Sample to < 10 ng/µL with PB elution buffer.
Add 3.7 x volume of Sample of dilute SPRI beads to the Sample and mix by flicking.
Place on a rotational mixer for 00:30:00 at Room temperature
Briefly spin the sample to collect and place on a magnetic rack, then allow the sample to clear.
Aspirate and retain the supernatant in a new 1.5 lo-bind tube.
Add 250 µL of 80 % volume EtOH across the beads and allow to sit for 00:00:30 .
30s
Remove and discard the EtOH without disturbing the beads.
Repeat the wash step and briefly spin to collect residual EtOH.
Remove residual EtOH then add 21 µL of 1x PB elution buffer to the beads and mix by flicking.
Incubate the Sample at 37 °C for 00:30:00 with occasional mixing then return to the magnetic rack.
30m
Dilute 1 µL of final library in 9 µL of molecular grade water and retain for QC.
Aspirate 20 µL of final library to a new 1.5 ml lo-bind tube and place on ice.
Store final library at 4 °C for imminent SMRT cell loading or a -20 °C for longterm storage.