Apr 28, 2022
Metagenomic extraction of high molecular weight plankton from filters
- 1Genoscope/CEA
Protocol Citation: Benoît Vacherie, Karine Labadie 2022. Metagenomic extraction of high molecular weight plankton from filters. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl89by8v2w/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: April 28, 2022
Last Modified: April 28, 2022
Protocol Integer ID: 61576
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Abstract
Filtration and extraction protocol of plankton metagenomic samples to obtain high molecular weight DNA for sequencing on Minion nanopore.
The protocol describes the following steps:
- filtration of the water on filters of different porosity
- Recovery and grinding of organisms
- HMW extraction
- Results
Guidelines
Use only wide-bore pipette tips
Materials
Reagent :
DNeasy PowerSoil Pro Kit (250)QiagenCatalog #Cat No./ID: 47014
Liquid nitrogen
NucleoProtect RNAMacherey-nagelCatalog #740400.500
1X PBS (Phosphate-buffered saline )
Nylon Net filter 20 µmMerck MilliporeCatalog #NY2004700
isopore membrane filter 3 µmMerck MilliporeCatalog #TSTP04700
Isopore membrane filter 0.2 µmMerck MilliporeCatalog #GTTP04700
Consumables :
2 mL Eppendorf
1.5ml Eppendorf DNA LoBind tubes
ART Wide-Bore tips 200µlCatalog #2069GPK
ART Wide-Bore tips 1000 µlCatalog #2079GPK
Equipment:
Equipment
new equipment
NAME
Perilstatic Pump
TYPE
Spectra Field Pro
BRAND
SFP-100
SKU
Equipment
new equipment
NAME
Horizontal Microtube Holder for 24 Tubes
BRAND
97060-178
SKU
Protocol materials
Isopore membrane filter 0.2 µmMerck Millipore (EMD Millipore)Catalog #GTTP04700
Materials, Step 3
DNeasy PowerSoil Pro Kit (250)QiagenCatalog #Cat No./ID: 47014
Materials, Step 7
1.5ml Eppendorf DNA LoBind tubes
Materials
ART Wide-Bore tips 200µlCatalog #2069GPK
Materials
ART Wide-Bore tips 1000 µlCatalog #2079GPK
Materials
NucleoProtect RNAMacherey-NagalCatalog #740400.500
Materials, Step 4
Nylon Net filter 20 µmMerck Millipore (EMD Millipore)Catalog #NY2004700
Materials, Step 3
isopore membrane filter 3 µmMerck Millipore (EMD Millipore)Catalog #TSTP04700
Materials, Step 3
2 mL Eppendorf
Materials
Liquid nitrogen
Materials, Step 4
Filtration
Filtration
1h
1h
From several liters of water (fresh or sea).
Cascade filtration on filters of different porosity with the help of a perilstatic pump.
Equipment
new equipment
NAME
Perilstatic Pump
TYPE
Spectra Field Pro
BRAND
SFP-100
SKU
Pre-filtration through a 100µm celular sieve to remove large debris and larger zooplanktonic organisms (not of interest in our case).
Equipment
celular sieve falcon 100 µm
NAME
celular sieve falcon 100 µm
BRAND
352360
SKU
Successive filtration on 3 filters of different porosities: 20 µm; 3 µm; 0.2 µm.
Nylon Net filter 20 µmVWR InternationalCatalog #NY2004700
isopore membrane filter 3 µmVWR InternationalCatalog #TSTP04700
Isopore membrane filter 0.2 µmVWR InternationalCatalog #GTTP04700
Filters are transferred into 5 ml tubes and flash frozen in liquid nitrogen.
The tubes are stored at -80°C before extraction.
Liquid nitrogenVWR International
Filters can be directly resuspended in nucléoprotect RNA and stored at 4°C.
NucleoProtect RNAVWR InternationalCatalog #740400.500
Recovery and grinding of organisms
Recovery and grinding of organisms
1h
1h
Add 3 ml of 1X PBS to the tube with the filter.
Vortex at maximum power to loosen the organisms from the membrane.
Remove the filter with sterile forceps.
1X PBS (Phosphate-buffered saline )VWR International
Centrifuge the tube to pellet the organisms.
5000 x g, Room temperature, 00:05:00
Gently remove the supernatant with a pipette.
5m
If the filters have been stored in Nucleoprotect, make a second wash in 500 µl of 1X PBS (same centrifugation)
Resuspend the pellet with 800 µl of CD1 buffer (PowerSoil kit).
DNeasy PowerSoil Pro Kit (250)VWR InternationalCatalog #Cat No./ID: 47014
Incubate 10 min at 65 °C with 300 rpm agitation.
300 rpm, 65°C, 00:10:00
Transfer the suspension to a powerbeads tube (PowerSoil kit).
Vortex at maximum speed for 3 x 1 min (with 1 min pause between each grinding cycle) using the horizontal tube adapter.
00:03:00
Equipment
new equipment
NAME
Horizontal Microtube Holder for 24 Tubes
BRAND
97060-178
SKU
3m
Transfer the grinding material by dipping the tip at the bottom of the tube into a 2ml tube.
Powersoil extraction
Powersoil extraction
30m
30m
Add 200 µl of CD2 solution (stored at 4°C) to the grinding and vortex 5 sec.
Centrifuge 1 min / 14 000 G / RT
14000 x g, Room temperature, 00:01:00
1m
Transfer the supernatant (avoiding touching the pellet) to a 2ml tube.
Add 600 µl of CD3 solution and vortex 5 sec.
Load 700 µl of the lysate on a MB spin column (PowerSoil kit)
Centrifuge 1 min / 14 000 G
14000 x g, Room temperature, 00:01:00
1m
Discard the filtrate and repeat step 13 until the lysate is consumed
Place the column on a new collection tube and add 500 µl of EA Solution
Centrifuge 1 min / 14,000 G
14000 x g, Room temperature, 00:01:00
1m
Discard the filtrate and add 500 µl of Solution C5
Centrifuge 1 min / 14,000 G
14000 x g, Room temperature, 00:01:00
1m
Place the column on a new collection tube
Centrifuge for 2 min / 14 000 G
14000 x g, Room temperature, 00:02:00
2m
Place the column on a 1.5 ml eppendorf tube
Add 25 µl of Solution C6 to the center of the column
Incubate 5 min at RT and Centrifuge 1 min / 14 000 G
00:05:00 Room temperature
14000 x g, Room temperature, 00:01:00
6m
Add 25 µl of Solution C6 to the center of the column
Incubate 5 min at RT and Centrifuge 1 min / 14,000 G
Remove the column.
00:05:00 Room temperature
14000 x g, Room temperature, 00:01:00
6m
Make the following quality controls:
- Qbit (concentration)
- Nanodrop (purity)
- Tapestation or Femto-pulse (molecule size estimation)
Store the tube at -20°C
Results
Results