TITLE: DNA extraction protocol for DNA-metabarcoding of marine phytoplankton using Zymobiomics DNA minprep kit (Zymo Research; D4300)
AUTHORS: Andersson    Agneta, Bengt   Karlson, Andersson    F. Anders, Sonia   Brugel, Latz    Meike, Lycken   Jenny, Mikael  Hedblom, Anders  Torstensson, Markus  Lindh

[DESCRIPTION]
The DNA extraction protocol described in this document follows the standard protocol provided with the Zymobiomics DNA miniprep kit (https://files.zymoresearch.com/protocols/_d4300t_d4300_d4304_zymobiomics_dna_miniprep_kit.pdf) with a few modifications. The protocol is part of the project "DNA-metabarcoding of marine phytoplankton" funded by the Swedish Environmental Protection Agency and the Swedish Agency for Marine and Water Management. 

Phytoplankton play a key role in global biogeochemical cycles and form the basis of marine food webs. Quantitative assessment of the abundance and biodiversity of phytoplankton is therefore a natural component of environmental monitoring programs. Until now such monitoring has largely been based on microscopic counting. This is a time consuming process and requires personnel with high skill for identifying the organisms, and the smallest cells can hardly be identified with this method. There is hence an urge for faster, more precise and reproducible ways of quantifying plankton species in environmental samples. High-throughput sequencing of taxonomic marker genes (metabarcoding) has emerged as a popular alternative for studying plankton diversity. However, also this method has its limitations, and in order to be robustly implemented in monitoring programs, protocols need to be optimised and validated. The main goal of the project is to evaluate high-throughput sequencing as a tool to investigate the diversity of phytoplankon in the seas surrounding Sweden, with special focus on non-indigenous species and harmful algae. Specifically we want to test effects of storage of samples, sample volume, replication and background DNA in the water (“eDNA”), develop and evaluate an improved method for identifying phytoplankton based on sequencing a longer region of the rRNA operon and compare results from metabarcoding with results from microscope-based methods. The tests will be conducted on water samples collected as part of the Swedish National Marine Monitoring programme. This will provide a large data set covering all major sea areas surrounding Sweden and allow us to compare the metabarcoding results with taxonomic identifications obtained using microscopy as part of the monitoring programme. This collaborative project will be conducted by a team of researchers with complementary expertise in plankton ecology, marine monitoring, molecular biology and bioinformatics.

[STEPS]
SECTION: Sample/Filter collection of biomass at sea
1. Filter water samples onto 0.2 µm pore size Millipore filter (Merck Millipore; GSWP04700) attached to a filter funnel connected to a vacuum source. Filter a minimum of 500 ml collected seawater. 

500 mL 


 

SECTION: Laboratory preparations
5. Clean your work bench with 70% ethanol.
Prepare sterilized forceps and petri dish on the bench.
Prepare pipettes in varying sizes with filter tips.
Take out beakers for discarding liquids and used tips.
Turn on the microcentrifuge and set the machine to 13000 x g for 1 minute.
Prepare Vortex with adapter plates and set timer for 10 minutes.
SECTION: Laboratory procedure
7. Thaw filters in the sampling tubes at room temperature and add the filter cut into small pieces to a ZR BashingBead™ Lysis Tube (0.1 & 0.5 mm). 

Room temperature 
 

SECTION: Sample/Filter collection of biomass at sea
2. Using two sets of sterile forceps, pick up the filter membrane at opposite edges and roll the filter into a 2 ml cryo tube (Avantor, 479-1262) with the top side of the filter facing inward. 
SECTION: Sample/Filter collection of biomass at sea
3. Take note of station, date, sample ID, sampling time, time duration of filtration, filtered volume and any comments of potential use (such as filtration problems, colour of filter etc.).
SECTION: Sample/Filter collection of biomass at sea
4. Store the filters at minimum -20°C (short-term storage) or -80°C (long-term storage) no additional reagent for preservation is needed.

-20 °C 
 

SECTION: Laboratory preparations
6. Prepare spike-in genes using artificial 16S and 18S rRNA gene oligonucelotides (designed in-house and generated by Twist Bioscience). See sequences and sub-steps below:
 


 


 

SECTION: Laboratory preparations
6.1. Add 100 µl sterile filtered autoclaved MQ, DNase/RNase Free Water or TE buffer to the dry pellet bringing the stock solution to a concentration of 10 ng/µl.

100 µL 

SECTION: Laboratory preparations
6.2. Then, in sequence through continued serial dilution in steps of x 10 dilutions, prepare working solutions with the final concentration of the 16S and 18S spike-in DNA to 0.0002 ng/µl  and 0.0007 ng/µl, respectively, note the x 10 dilution for the final concentration compared to Table 2.
SECTION: Laboratory preparations
6.3. Aliquot and freeze working solution in batches at -20°C until processing. 

-20 °C 

SECTION: Laboratory procedure
8. Rinse the sampling tube with 750 µl and add the Lysis Solution into the ZR BashingBead™ Lysis Tube. 

 750 µL 

Note: For samples stored and lysed in DNA/RNA Shield™ Lysis buffer, do not add ZymoBIOMICS™ Lysis Solution and proceed to step 9.


SECTION: Laboratory procedure
9. Add 10 µl of the prepared dilutions of 16S and 18S spikes, and cap tightly. 

10 µL 

SECTION: Laboratory procedure
10. Secure in a Vortex-Genie 2 fitted with a 2 ml tube holder assembly (adapter) and process at maximum speed for 10 minutes. 

  

Note: Vortex for 10 min at maximum speed (2850Hz) on a Vortex-Genie 2 is a specific selected step and slight modification from the standard ZymoBIOMICS protocol.
 


SECTION: Laboratory procedure
11. Centrifuge the ZR BashingBead™ Lysis Tubes (0.1 & 0.5 mm) in a microcentrifuge at 13,000 x g for 1 minute.

13000 x g, 1 min 
 

SECTION: Laboratory procedure
12. Transfer up to 400 µl supernatant to the Zymo-Spin™ III-F Filter (red mark) in a Collection Tube and centrifuge at 8,000 x g for 1 minute. Discard the Zymo-Spin™ III-F Filter.

400 µL 

8000 x g, 1 min 
 


SECTION: Laboratory procedure
13. Binding preparation: Add 1,200 µl of ZymoBIOMICS™ DNA Binding Buffer to the filtrate in the Collection Tube from Step 12. Mix well.

1200 µL 





SECTION: Laboratory procedure
14. Transfer 800 µl of the mixture from Step 13 to a Zymo-Spin™ IICR Column in a Collection Tube and centrifuge at 10,000 x g for 1 minute. 

10000 x g, 1 min 
 

SECTION: Laboratory procedure
15. Discard the flow through from the Collection Tube and REPEAT STEP 14.
SECTION: Laboratory procedure
16. Place the Zymo-Spin™ IICR Column in a NEW Collection Tube.
SECTION: Laboratory procedure
17. Add 400 µl ZymoBIOMICS™ DNA Wash Buffer 1 to the Zymo-Spin™ IICR Column and centrifuge at 10,000 x g for 1 minute. Discard the flow-through.

400 µL 

10000 x g, 1 min 


 


SECTION: Laboratory procedure
18. Add 700 µl ZymoBIOMICS™ DNA Wash Buffer 2 to the Zymo-Spin™ IICR Column in a Collection Tube and centrifuge at 10,000 x g for 1 minute. Discard the flow-through.

700 µL 

10000 x g, 1 min 


 


SECTION: Laboratory procedure
19. Add 200 µl ZymoBIOMICS™ DNA Wash Buffer 2 to the Zymo-Spin™ IICR Column in a Collection Tube and centrifuge at 10,000 x g for 1 minute.

200 µL 

10000 x g, 1 min 


 


SECTION: Laboratory procedure
20. Transfer the Zymo-Spin™ IICR Column to a clean 1.5 ml microcentrifuge tube and add 50 µl ZymoBIOMICS™ DNase/RNase Free Water directly to the center of the column matrix and incubate in room temperature for 1 minute. Centrifuge at 10,000 x g for 1 minute to elute the DNA.

50 µL 

Room temperature 

10000 x g, 1 min 

Note: Adding 50 μl of ZymoBIOMICS DNase/RNase Free Water is a specific selected step and slight modification from the standard ZymoBIOMICS protocol.
 


SECTION: Laboratory procedure
21. Place a Zymo-Spin™ III-HRC Filter in a NEW Collection Tube and add 600 µl ZymoBIOMICS™ HRC Prep Solution. Centrifuge at 8,000 x g for 3 minutes.

600 µL 

8000 x g, 3 min 

SECTION: Laboratory procedure
22. Place the prepared Zymo-Spin™ III-HRC Filter in a clean 1.5 ml microcentrifuge tube. Make sure to mark the tube (both lid and side) clearly with a unique ID using a permanent marker resistant to freezing.
SECTION: Laboratory procedure
23. Transfer the eluted DNA (Step 20) to the Zymo-Spin™ III-HRC Filter and centrifuge at exactly 16,000 x g for 3 minutes.

16000 x g, 3 min 


 

SECTION: Laboratory procedure
24. Freeze extracted gDNA at -20°C or -80°C. Measure the concentration of the extracted DNA using Qubit Fluorometer; see separate protocol for Qubit provided by the manufacturer. The filtered DNA is now suitable for PCR and other downstream applications.

-20 °C