TITLE: Quantifying ecosystem service provider interactions via bulk sample DNA metabarcoding
AUTHORS: Jordan P Cuff, James JN Kitson, darren.evans

[DESCRIPTION]
This protocol is designed for extracting DNA from pan trap-collected invertebrates for biomonitoring and community metabarcoding to identify and quantify ecosystem service and disservice providers. The reagents and methods proposed offer a cost effective and high-throughput method for molecular diversity analyses of bulk samples using standard lab equipment.

[STEPS]
SECTION: Preparation and homogenisation of samples
15. Vortex each sample to mix and incubate at 37 °C overnight (12-16 hours).
SECTION: DNA extraction and purification
20. Add all of the sample solution (~ 600 μL) to a well in a 96-well silica membrane spin-column plate and cover with a breathable seal. 
 

SECTION: DNA extraction and purification
21. Centrifuge at ≥ 6,000 x g for 10 minutes and discard the flow-through.
 

SECTION: DNA extraction and purification
22. Add 500 μL Wash Buffer 1 to each spin column and cover with a breathable seal.
SECTION: DNA extraction and purification
22.1. Wash Buffer 1 should be comprised of the following reagents:

 

SECTION: DNA extraction and purification
23. Centrifuge at ≥ 6,000 x g for 5 minutes and discard the flow-through.
 

SECTION: DNA extraction and purification
24. Add 500 μL Wash Buffer 2 to each spin column and cover with a breathable seal.
SECTION: DNA extraction and purification
24.1. Wash Buffer 2 should be pH ~7 and comprised of the following reagents:

 

SECTION: DNA extraction and purification
25. Centrifuge at ≥ 6,000 x g for 15 minutes and discard the flow-through.
 

SECTION: DNA extraction and purification
26. Carefully move the spin column plate to a new 0.5 mL DNA collection plate.
 

SECTION: DNA extraction and purification
27. Add 200 μL Elution Buffer directly to the silica membrane and leave it at room temperature for 5 minutes, covering with a breathable seal.
SECTION: DNA extraction and purification
27.1. Elution Buffer should be pH ~7 and comprised of the following reagents:
 
 

SECTION: DNA extraction and purification
28. Centrifuge at ≥ 6000 x g for 2 minutes. The DNA is now in the collection plate and can be taken forward to molecular analysis. 
 

SECTION: Trapping of invertebrates
1. Select suitable sites and locations for bulk sample traps (e.g., pan traps). Consider how systematic the study needs to be and the various constraints imposed on the data by the study design. The following steps will focus on the example of water pan traps.
SECTION: Preparation and homogenisation of samples
14. To the 1 mL lysate, add 500 μL of freshly-prepared Proteinase Buffer, a master mix of Lysis Buffer 2 and proteinase K (detailed in the sub-steps below) and vortex to mix.
SECTION: Preparation and homogenisation of samples
14.1. Lysis Buffer 2 should be pH 9 and comprised of the following reagents:
 
 

SECTION: Preparation and homogenisation of samples
14.2. Per plate, the Proteinase Buffer master mix should comprise:

 

SECTION: Preparation and homogenisation of samples
16. Centrifuge at 4,000 x g for 4 minutes.
SECTION: Preparation and homogenisation of samples
17. Transfer 1 mL of the supernatant to a clean 2.2 mL deep-well 96-well plate for archiving/backup.
SECTION: Preparation and homogenisation of samples
18. Transfer 200 μL of the supernatant to a clean 2.2 mL deep-well 96-well plate. 
 

SECTION: DNA extraction and purification
19. Add 400 μL of master mix of Protein Denaturation Buffer and ethanol (detailed in the sub-step below)  to each well of the 2.2 mL 96-well plate.
SECTION: DNA extraction and purification
19.1. Protein Denaturation Buffer should be comprised of the following reagents:
  
 

SECTION: DNA extraction and purification
19.2. Per 96-well plate, the Protein Denaturation Buffer and ethanol master mix should comprise:

 

SECTION: DNA extraction: purification
None. Add 200 μL of the supernatant from STEP 10 into each well containing Protein Denaturation Buffer and ethanol. 
SECTION: DNA extraction: purification
None. Per 96-well plate, the Protein Denaturation Buffer and ethanol master mix should comprise:

 

SECTION: DNA extraction: purification
None. Add all of the sample solution (~ 600 μL) to a well in a 96-well DNA membrane spin column and cover with a breathable seal. 
 

SECTION: DNA extraction: purification
None. Centrifuge at ≥ 6000 x g for 1 minute and discard the flow-through.
 

SECTION: DNA extraction: purification
None. Add 500 μL Wash Buffer 1 to each spin column and cover with a breathable seal.
SECTION: DNA extraction: purification
None. Centrifuge at ≥ 6000 x g for 1 minute and discard the flow-through.
 

SECTION: DNA extraction: purification
None. Wash Buffer 1 should be comprised of the following reagents:

 

SECTION: DNA extraction: purification
None. Add 500 μL Wash Buffer 2 to each spin column and cover with a breathable seal.
SECTION: DNA extraction: purification
None. Wash Buffer 2 should be pH ~7 and comprised of the following reagents:

 

SECTION: DNA extraction: purification
None. Centrifuge at 20,000 x g for 3 minutes and discard the flow-through.
 

SECTION: DNA extraction: purification
None. Carefully move the spin column plate to a new 0.5 mL DNA collection plate.
 

SECTION: DNA extraction: purification
None. Add 200 μL Elution Buffer directly to the silica membrane and leave it at room temperature for 5 minutes, covering with a breathable seal.
SECTION: DNA extraction: purification
None. Elution Buffer should be pH ~7 and comprised of the following reagents:
 
 

SECTION: DNA extraction: purification
None. Centrifuge at ≥ 6000 x g for 1 minute. The DNA is now in the collection plate and can be taken forward to amplification. 
 

SECTION: DNA extraction: purification
None. Add 400 μL of master mix of Protein Denaturation Buffer and ethanol (detailed below) to each well of the 2.2 ml 96-well plate.
SECTION: DNA extraction: purification
None. Protein Denaturation Buffer should be comprised of the following reagents:
  
 

SECTION: DNA extraction: purification
None. Per 96-well plate, the Protein Denaturation Buffer and ethanol master mix should comprise:

 

SECTION: DNA extraction: purification
None. Add all of the sample solution (~ 600 μL) to a well in a 96-well silica membrane spin column and cover with a breathable seal. 
 

SECTION: DNA extraction: purification
None. Centrifuge at ≥ 6000 x g for 10 minutes and discard the flow-through.
 

SECTION: DNA extraction: purification
None. Add 500 μL Wash Buffer 1 to each spin column and cover with a breathable seal.
SECTION: DNA extraction: purification
None. Wash Buffer 1 should be comprised of the following reagents:

 

SECTION: DNA extraction: purification
None. Centrifuge at ≥ 6000 x g for 5 minutes and discard the flow-through.
 

SECTION: DNA extraction: purification
None. Add 500 μL Wash Buffer 2 to each spin column and cover with a breathable seal.
SECTION: DNA extraction: purification
None. Carefully move the spin column plate to a new 0.5 mL DNA collection plate.
 

SECTION: DNA extraction: purification
None. Wash Buffer 2 should be pH ~7 and comprised of the following reagents:
 
 

SECTION: DNA extraction: purification
None. Centrifuge at ≥ 6000 x g for 15 minutes and discard the flow-through.
 

SECTION: DNA extraction: purification
None. Add 200 μL Elution Buffer directly to the silica membrane and leave it at room temperature for 5 minutes, covering with a breathable seal.
SECTION: DNA extraction: purification
None. Elution Buffer should be pH ~7 and comprised of the following reagents:
 
 

SECTION: DNA extraction: purification
None. Centrifuge at ≥ 6000 x g for 2 minutes. The DNA is now in the collection plate and can be taken forward to amplification. 
 

SECTION: Initial digestion of OPM larvae
None. Grind the larvae in a Geno/Grinder 2010 at full speed (1750 RPM) for 4 minutes.
 

SECTION: Initial digestion of OPM larvae
None. Centrifuge at 4,000 g for 2 minutes.
SECTION: Initial digestion of OPM larvae
None. Remove the ball bearings using a magnet, taking care not to cross-contaminate the samples.
SECTION: Initial digestion of OPM larvae
None. To the lysate, add 1 mL of freshly-prepared Proteinase Buffer master mix of Lysis Buffer 2 and proteinase K (detailed in the sub-steps below) and vortex to mix.
SECTION: Initial digestion of OPM larvae
None. Lysis Buffer 2 should be pH 9 and comprised of the following reagents:
 
 

SECTION: Initial digestion of OPM larvae
None. Per sample, the Proteinase Buffer master mix should comprise:

 

SECTION: Initial digestion of OPM larvae
None. Incubate at 37 °C overnight (12-16 hours)
 

SECTION: Initial digestion of OPM larvae
None. Centrifuge at 4000 x g for 4 minutes.
SECTION: Initial digestion of OPM larvae
None. Transfer 1 mL of the supernatant to a clean 2.2 mL deep-well 96-well plate for archiving/backup and retain the rest in the original plate.
SECTION: Initial digestion of OPM larvae
None. Centrifuge at 2000 x g for 4 minutes.
SECTION: Initial digestion of OPM larvae
None. Transfer 200 μL of the supernatant to a clean 2.2 mL deep-well 96-well plate. 
 

SECTION: Preparation and homogenisation of samples
6. Add three 3 mm hardened carbon steel beads to the tube.
 

SECTION: Preparation and homogenisation of samples
9. Two lysis options are presented below. One standard protocol using Proteinase K to lyse a subsample of each trap (this may reduce detectable diversity but increases efficiency), and the other uses papain (a cheap alternative to Proteinase K) to lyse whole samples.
SECTION: Trapping of invertebrates
2. Set a pan trap at approximately the same height as the surrounding vegetation. 
 

SECTION: Trapping of invertebrates
3. Fill the traps with water containing detergent to reduce surface tension. Leave the traps out for 72 hours.
 

SECTION: Trapping of invertebrates
4. Pour the trap contents through a sterilised funnel into a suitably sized pot labelled with all necessary sample information for transport to the laboratory. Store samples at -20 °C until ready to process.
SECTION: Preparation and homogenisation of samples
8. Directly from the freezer, grind the bulk samples in a tissue grinder/homogeniser/lyser at 1750 RPM for 4 minutes.
 

SECTION: Preparation and homogenisation of samples
13. Take a subsample of 1 mL lysate from the supernatant and transfer it to a 2.2 mL 96-well plate. Store the remaining lysate and sample from the falcon tube at -20 °C.
 

SECTION: Preparation and homogenisation of samples
11. Vortex each sample to mix and incubate at 37 °C overnight (12-16 hours).
SECTION: Preparation and homogenisation of samples
12. Vortex to mix and centrifuge at 4,000 x g for 4 minutes.
SECTION: Preparation and homogenisation of samples
12. Centrifuge at 4,000 x g for 2 minutes.
SECTION: Preparation and homogenisation of samples
13. Transfer 1 mL of the supernatant to a clean 2.2 mL deep-well 96-well plate for archiving/backup.
SECTION: DNA extraction and purification
15. Add 400 μL of master mix of Protein Denaturation Buffer and ethanol (detailed in the sub-step below)  to each well of the 2.2 mL 96-well plate.
SECTION: Preparation and homogenisation of samples
14. Transfer 200 μL of the supernatant to a clean 2.2 mL deep-well 96-well plate. 
 

SECTION: DNA extraction and purification
16. Add all of the sample solution (~ 600 μL) to a well in a 96-well silica membrane spin-column plate and cover with a breathable seal. 
 

SECTION: DNA extraction and purification
15.1. Protein Denaturation Buffer should be comprised of the following reagents:
 
 

SECTION: DNA extraction and purification
15.2. Per 96-well plate, the Protein Denaturation Buffer and ethanol master mix should comprise:
 
 

SECTION: DNA extraction and purification
17. Centrifuge at ≥ 6,000 x g for 10 minutes and discard the flow-through.
 

SECTION: DNA extraction and purification
18. Add 500 μL Wash Buffer 1 to each spin column and cover with a breathable seal.
SECTION: DNA extraction and purification
18.1. Wash Buffer 1 should be comprised of the following reagents:

 

SECTION: DNA extraction and purification
19. Centrifuge at ≥ 6,000 x g for 5 minutes and discard the flow-through.
 

SECTION: DNA extraction and purification
20. Add 500 μL Wash Buffer 2 to each spin column and cover with a breathable seal.
SECTION: DNA extraction and purification
20.1. Wash Buffer 2 should be pH ~7 and comprised of the following reagents:

 

SECTION: DNA extraction and purification
21. Centrifuge at ≥ 6,000 x g for 15 minutes and discard the flow-through.
 

SECTION: DNA extraction and purification
22. Carefully move the spin column plate to a new 0.5 mL DNA collection plate.
 

SECTION: DNA extraction and purification
23. Add 200 μL Elution Buffer directly to the silica membrane and leave it at room temperature for 5 minutes, covering with a breathable seal.
SECTION: DNA extraction and purification
23.1. Elution Buffer should be pH ~7 and comprised of the following reagents:
 
 

SECTION: DNA extraction and purification
24. Centrifuge at ≥ 6000 x g for 2 minutes. The DNA is now in the collection plate and can be taken forward to molecular analysis. 
 

SECTION: Preparation and homogenisation of samples
5. Wash samples with water in a fine mesh sieve to remove external contaminants, and transfer to a 50 mL falcon tube.

 

SECTION: Preparation and homogenisation of samples
7. Freeze the samples overnight at -80 °C.
SECTION: Preparation and homogenisation of samples
10. Add an appropriate volume of fresh Lysis Buffer 1 (detailed in the sub-step below) to sufficiently cover the specimens.
 

SECTION: Preparation and homogenisation of samples
10.1. Lysis Buffer 1 should be pH 9 and comprised of the following reagents:

 

SECTION: Preparation and homogenisation of samples
10. To each sample, add 30 mL of fresh Lysis Buffer 1 and 15 mL of Proteinase Buffer (a master mix of Lysis Buffer 2 and papain; all reagents detailed in the sub-steps below).
 

 

SECTION: Preparation and homogenisation of samples
10.1. Lysis Buffer 1 should be pH 9 and comprised of the following reagents:

 

SECTION: Preparation and homogenisation of samples
10.2. Lysis Buffer 2 should be pH 9 and comprised of the following reagents:

 

SECTION: Preparation and homogenisation of samples
10.3. Per sample, the Proteinase Buffer master mix should comprise:
 
 

SECTION: Preparation and homogenisation of samples
11. Grind the samples in a Geno/Grinder at full speed (1750 RPM) for 2 minutes.
SECTION: DNA amplification
29. Decide how samples will be distributed across plates (but don't distribute DNA yet). Consider including a negative control in each row and column to detect any contaminants in each tagged forward and reverse primer. Among these wells, include any DNA extraction negative controls. Include positive controls (ideally mixed samples of species not found in the same study system), perhaps one adjacent to negative controls and the other adjacent only to samples (but both on separate rows and columns). Include blank controls (ideally wells into which no reagents or at least no primers are added), perhaps one adjacent to negative controls and the other adjacent only to samples (but both on separate rows and columns).

If using multiple PCR primer pairs, familiarise yourself with the annealing temperatures for each and prepare separate PCR plates for each. For optimal accuracy, consider running replicates of each reaction (e.g., triplicates).

 
38. Prepare 0.1X SPRI bead solution and bring to room temperature. The below steps detail how to make this solution, but it is also commercially available.
SECTION: DNA library preparation and sequencing
59. Quantify the concentration of the library from the previous step (e.g., using Qubit dsDNA assay).
SECTION: Bioinformatics
69. Optimal bioinformatics workflows depend on a variety of factors including target taxa, study system, the genetic marker used, computational resources available and various other factors. Other resources with code integration will be much better suited to detailing the steps involved. The process will likely follow a similar progression of steps though:
Read processing: demultiplex, trim primers, merge pairs.
Filtering and error correction: quality filtering, dereplication, denoising, chimera filtering.
Taxonomic unit aggregation: OTU/ASV delimitation.
Taxon assignment: reference database curation, taxon assignment.
SECTION: Ecosystem service provider quantification
70. Following taxon assignment to ASVs, taxa must be assigned as ecosystem service providers, ecosystem disservice providers, or neutral taxa. For an example, see the supplementary information in Cuff et al. (2022).
 

SECTION: Ecosystem service provider quantification
71. Ecosystem service and disservice providers can then be summed for each sample, giving quantities of each which can be analysed against sample metadata.
SECTION: DNA amplification
30. Distribute one drop of mineral oil into each well of the PCR plate (~20 µL). 


 

SECTION: DNA amplification
31. Prepare enough PCR mastermix for each sample.

For a full plate, these values will usually suffice (with some overage to account for pipetting error), but check your specific Taq polymerase mix for any differences:
        Volume

 

SECTION: DNA amplification
32. For ease, if able to use a 96-well pipette, consider creating a "primer plate" containing both PCR primers for each well at 5 µM concentration; this is especially effective when using multiple plates. Add 7.75 µL mastermix to each well, followed by 1.25 µL of each primer mix to its corresponding well. It is possible to use this strategy with a multi-channel or even single-channel pipette, but the suggestion immediately below may be easier. 

If not using a 96-well pipette, consider making eight mastermixes each with sufficient reagents for 12 samples (7.75 µL mastermix per well, so 97.65 µL for 12 with some overage for pipetting error), each containing a different forward primer (with 0.625 µL of 5 µM forward primer per well, so 7.875 µL per mastermix for 12 with some overage for pipetting error). Distribute 8.375 µL of forward primer + mastermix to each well across the rows corresponding to each forward primer. Then distribute 0.625 µL of reverse primer to each well in the corresponding columns making sure to change pipette tip for each sample to avoid cross-contaminating the forward primers.

 
SECTION: DNA amplification
33. Add 1 µL DNA to each corresponding sample and positive control well, and 1 µL molecular grade water to each negative control other than extraction negative controls (which will be added as samples).
SECTION: DNA amplification
34. Briefly centrifuge the plate to ensure that the oil is above the PCR mix and everything is at the bottom of each well without air bubbles.
SECTION: DNA amplification
35. Load the PCR plate into a thermocycler. Ensure that the temperature regime matches the enzyme used (including any heat activation for hot-start Taq) and that the annealing temperature matches the PCR primers used.

 

41. Incubate at RT for 5 mins.
42. Place on a magnetic stand for 5 mins.
40. Add 20 µL of PCR product (or whatever volume of beads was used in the last step) to each corresponding well of 0.1X beads, avoiding oil, and mix by vortexing (1500 RPM for 1 min).

 

38.1. If using beads such as Sera-Mag Magnetic SpeedBeads (carboxylated, 1 µm, 3 EDAC/PA5), take 1 mL of well-mixed bead solution and wash the beads twice with TE+Tween buffer (10 mM Tris base, 1 mM EDTA, 0.05% Tween 20, pH 8.0) by magnetising the beads, removing the supernatant, adding the TE+Tween, remagnetising the beads and removing the supernatant, and repeating the addition and removal of TE+Tween once more.
38.2. To the beads, add the following mix:
 

38.3. Add 20 mL of 50 % PEG to the tube to reach a 1X bead solution (alongside making the 0.1X solution, this will be useful later).
43. Remove all but 5 µL of the mixture from each well via pipette without disturbing the beads, which should be settled on the magnet (although can be hard to see with 0.1X bead solutions).
44. Add 200 µL 80 % ethanol to each well.
45. Remove the ethanol and add a further 200 µL 80 % ethanol to each well.
46. Remove the ethanol as completely as possible by pipetting.

 

47. Add 10 µL molecular grade water to each well, shake at 1500 RPM for 1 min and incubate for 5 mins.
48. Place on a magnetic stand for 5 mins.
49. Pool 8 µL from each well into a single tube, leaving magnetic beads behind in the 96-well plate.

 

50. Mix the pooled samples by flicking and inverting, and transfer 700 µL into a new 2.5 mL microcentrifuge tube.
51. Add 700 µL 1X magnetic beads for DNA purification (see note below for size selection) and mix by vortexing. Incubate for 5 mins.


 

SECTION: DNA amplification
36. The samples should now be checked for successful amplification, contamination in negatives and any secondary banding. Gel electrophoresis will achieve this, but digital systems like the Qiagen Qiaxcel will do this and facilitate equalisation by generating amplicon-specific DNA concentations.
52. Place on a magnetic stand for 5 mins.
53. Add 400 µL 80 % ethanol.
54. Remove the ethanol and add a further 400 µL 80 % ethanol.
55. Remove the ethanol completely. Briefly centrifuge the sample to collect and remove any residual ethanol. Allow to air-dry until the aggregation of magnetic beads transitions from 'glossy' (shiny reflection of light) to 'matte' (dull dark brown mass), but not so long that it dries completely (i.e., begins to turn a rusty red and shows cracks).
56. Add 100 µL molecular grade water and mix by vortexing. Incubate for 5 mins.
57. Place the sample on a magnetic stand for 5 mins.
58. Remove 95 µL of the water and place it in a new tube. This will be the library used in the subsequent section.
SECTION: DNA library preparation and sequencing
60. Dilute library to ≤5 ng/µL in molecular grade water.

 

SECTION: DNA library preparation and sequencing
61. Add a drop of mineral oil to one PCR tube for each library, alongside an additional tube for a negative control.
SECTION: DNA library preparation and sequencing
62. Per library, assemble the following reaction:
 

SECTION: DNA library preparation and sequencing
63. Briefly centrifuge the tubes to ensure that the oil is above the PCR mix.
SECTION: DNA library preparation and sequencing
64. Load the PCR reactions into a thermocycler. Ensure that the temperature regime matches the enzyme used (including any heat activation for hot-start Taq) and that the annealing temperature matches the PCR primers used. Run for 12 cycles.
SECTION: DNA library preparation and sequencing
65. Quantify the concentration of the library from the previous step (e.g., using Qubit dsDNA assay).
SECTION: DNA library preparation and sequencing
66. Check that the adapters have been added to the DNA by determining and comparing the amplicon sizes for libraries before and after this second PCR. Amplicons should be longer after the second PCR. This is ideally assessed using a digital system like TapeStation, but even gel electrophoresis will work.
SECTION: DNA library preparation and sequencing
67. Once successful adapter addition is confirmed, libraries can be pooled so that each is equimolar in the final mixture based on the concentrations determined above (e.g., by Qubit).

 

SECTION: DNA library preparation and sequencing
68. Check with your sequencing provider how many fmol they will need in how many µL. This will be based on the sequencer, sequencing cartridge and any QC processes they follow. The libraries should be ready for sequencing.
SECTION: DNA normalisation
37. If the PCRs were replicated (i.e., each sample run multiple times for each PCR primer pair used), these can be merged together into one plate at this point, or carried forward separately. Keeping the replicates separate increases the number of libraries to prepare and sequence later, but better facilitates identification of inconsistencies between samples. To merge triplicates, assuming use of 10 µL reaction volumes, pipette 8 µL from each well of two of the three plates into the corresponding well of the third. Briefly centrifuge the merged plate to move the oil to the top of the product again.

 

38.4. Add 5 mL of 1X bead solution to 45 mL of the following mix to make a 0.1X solution:
 

39. If ≥20 µL of PCR product is available in each well, pipette 20 µL of 0.1X SPRI bead solution into each well of a new 96-well plate. If less DNA is available, add the available volume of 0.1X SPRI beads and add the same volume of PCR product to these beads in the next step.

 

SECTION: DNA amplification
25. Decide how samples will be distributed across plates (but don't distribute the DNA yet). Consider including a negative control in each row and column to detect any contaminants in each tagged forward and reverse primer. Among these wells, include any DNA extraction negative controls. Include positive controls (ideally mixed samples of species not found in the same study system), perhaps one adjacent to negative controls and the other adjacent only to samples (but both on separate rows and columns). Include blank controls (ideally wells into which no reagents or at least no primers are added), perhaps one adjacent to negative controls and the other adjacent only to samples (but both on separate rows and columns).

If using multiple PCR primer pairs, familiarise yourself with the annealing temperatures for each and prepare separate PCR plates for each. For optimal accuracy, consider running replicates of each reaction (e.g., triplicates).

 
SECTION: DNA amplification
26. Distribute one drop of mineral oil into each well of the PCR plate (~20 µL). 


 

SECTION: DNA amplification
27. Prepare enough PCR mastermix for each sample.

For a full plate, these values will usually suffice (with some overage to account for pipetting error), but check your specific Taq polymerase mix for any differences:
        Volume

 

SECTION: DNA amplification
28. For ease, if able to use a 96-well pipette, consider creating a "primer plate" containing both PCR primers for each well at 5 µM concentration; this is especially effective when using multiple plates. Add 7.75 µL mastermix to each well, followed by 1.25 µL of each primer mix to its corresponding well. It is possible to use this strategy with a multichannel pipette, but the next suggestion may be easier. 

If not using a 96-well pipette, consider making eight mastermixes each with sufficient reagents for 12 samples (7.75 µL mastermix per well, so 97.65 µL for 12 with some overage for pipetting error), each containing a different forward primer (with 0.625 µL of 5 µM forward primer per well, so 7.875 µL per mastermix for 12 with some overage for pipetting error). Distribute 8.375 µL of forward primer + mastermix to each well across the rows corresponding to each forward primer. Then distribute 0.625 µL of reverse primer to each well in the corresponding columns making sure to change pipette tip for each sample to avoid cross-contaminating the forward primers.

 
SECTION: DNA amplification
29. Add 1 µL DNA to each corresponding sample or positive control well, and 1 µL molecular grade water to each negative control other than extraction negative control(s).
SECTION: DNA amplification
30. Briefly centrifuge the plate to ensure that the oil is above the PCR mix and everything is at the bottom of each well without air bubbles.
SECTION: DNA amplification
31. Load the PCR plate into a thermocycler. Ensure that the temperature regime matches the enzyme used (including any heat activation for hot-start Taq) and that the annealing temperature matches the PCR primers used.

 

SECTION: DNA amplification
32. The samples should now be checked for successful amplification, contamination in negatives and any secondary banding. Gel electrophoresis will achieve this, but digital systems like the Qiagen Qiaxcel will do this and facilitate equalisation by generating amplicon-specific DNA concentations.
SECTION: DNA normalisation
33. If the PCRs were replicated (i.e., each sample run multiple times for each PCR primer pair used), these can be merged together into one plate at this point, or carried forward separately. Keeping the replicates separate increases the number of libraries to prepare and sequence later, but better facilitates inconsistencies between samples. To merge triplicates, assuming use of 10 µL reaction volumes, pipette 8 µL from each well of two of the three plates into the corresponding well of the third. Briefly centrifuge the merged plate to move the oil to the top of the product again.

 

SECTION: DNA normalisation
34. Prepare 0.1X SPRI bead solution and bring to room temperature. The below steps detail how to make this solution, but it is also commercially available.
SECTION: DNA normalisation
35. If ≥20 µL of PCR product is available in each well, pipette 20 µL of 0.1X SPRI bead solution into each well of a new 96-well plate. If less DNA is available, add the available volume of 0.1X SPRI beads and add the same volume of PCR product to these beads in the next step.

 

SECTION: DNA normalisation
34.1. If using beads such as Sera-Mag Magnetic SpeedBeads (carboxylated, 1 µm, 3 EDAC/PA5), take 1 mL of well-mixed bead solution and wash the beads twice with TE+Tween buffer (10 mM Tris base, 1 mM EDTA, 0.05% Tween 20, pH 8.0) by magnetising the beads, removing the supernatant, adding the TE+Tween, remagnetising the beads and removing the supernatant, and repeating the addition and removal of TE+Tween once more.
SECTION: DNA normalisation
34.2. To the beads, add the following mix:
 

SECTION: DNA normalisation
34.3. Add 20 mL of 50 % PEG to the tube to reach a 1X bead solution (alongside making the 0.1X solution, this will be useful later).
SECTION: DNA normalisation
34.4. Add 5 mL of 1X bead solution to 45 mL of the following mix to make a 0.1X solution:
 

SECTION: DNA normalisation
36. Add 20 µL of PCR product (or whatever volume of beads was used in the last step) to each corresponding well of 0.1X beads, avoiding oil, and mix by vortexing (1500 RPM for 1 min).

 

SECTION: DNA normalisation
37. Incubate at RT for 5 mins.
SECTION: DNA normalisation
38. Place on a magnetic stand for 5 mins.
SECTION: DNA normalisation
39. Remove all but 5 uL of the mixture from each well via pipette without disturbing the beads, which should be settled on the magnet (although can be hard to see with 0.1X solutions).
SECTION: DNA normalisation
40. Add 200 µL 80 % ethanol to each well.
SECTION: DNA normalisation
41. Remove the ethanol and add a further 200 µL 80 % ethanol to each well.
SECTION: DNA normalisation
42. Remove the ethanol as completely as possible by pipetting.

 

SECTION: DNA normalisation
43. Add 10 µL molecular grade water to each well, shake at 1500 RPM for 1 min and incubate for 5 mins.
SECTION: DNA normalisation
44. Place on a magnetic stand for 5 mins.
SECTION: DNA normalisation
45. Pool 8 µL from each well into a single tube, leaving magnetic beads behind in the 96-well plate.

 

SECTION: DNA normalisation
46. Mix the pooled samples by flicking and inverting, and transfer 700 µL into a new 2.5 mL microcentrifuge tube.
SECTION: DNA normalisation
47. Add 700 µL 1X magnetic beads for DNA purification (see note below for size selection) and mix by vortexing. Incubate for 5 mins.

 

SECTION: DNA normalisation
48. Place on a magnetic stand for 5 mins.
SECTION: DNA normalisation
49. Add 400 µL 80 % ethanol.
SECTION: DNA normalisation
50. Remove the ethanol and add a further 400 µL 80 % ethanol.
SECTION: DNA normalisation
51. Remove the ethanol completely. Briefly centrifuge the sample to collect and remove any residual ethanol. Allow to air-dry until the aggregation of magnetic beads transitions from 'glossy' (shiny reflection of light) to 'matte' (dull dark brown mass), but not so long that it dries completely (i.e., begins to turn a rusty red and shows cracks).
SECTION: DNA normalisation
52. Add 100 µL molecular grade water and mix by vortexing. Incubate for 5 mins.
SECTION: DNA normalisation
53. Place the sample on a magnetic stand for 5 mins.
SECTION: DNA normalisation
54. Remove 95 µL of the water and place it in a new tube. This will be the library used in the subsequent section.
SECTION: DNA library preparation and sequencing
55. Quantify the concentration of the library from the previous step (e.g., using Qubit dsDNA assay).
SECTION: DNA library preparation and sequencing
56. Dilute library to ≤5 ng/µL in molecular grade water.

 

SECTION: DNA library preparation and sequencing
57. Add a drop of mineral oil to one PCR tube for each library, alongside an additional tube for a negative control.
SECTION: DNA library preparation and sequencing
58. Per library, assemble the following reaction:
 

 
SECTION: DNA library preparation and sequencing
59. Briefly centrifuge the tubes to ensure that the oil is above the PCR mix.
SECTION: DNA library preparation and sequencing
60. Load the PCR reactions into a thermocycler. Ensure that the temperature regime matches the enzyme used (including any heat activation for hot-start Taq) and that the annealing temperature matches the PCR primers used. Run for 12 cycles.
SECTION: DNA library preparation and sequencing
61. Quantify the concentration of the library from the previous step (e.g., using Qubit dsDNA assay).
SECTION: DNA library preparation and sequencing
62. Check that the adapters have been added to the DNA by determining and comparing the amplicon sizes for libraries before and after this second PCR. Amplicons should be longer after the second PCR. This is ideally assessed using a digital system like TapeStation, but even gel electrophoresis will work.
SECTION: DNA library preparation and sequencing
63. Once successful adapter addition is confirmed, libraries can be pooled so that each is equimolar in the final mixture based on the concentrations determined above (e.g., by Qubit).

 

SECTION: DNA library preparation and sequencing
64. Check with your sequencing provider how many fmol they will need in how many µL. This will be based on the sequencer, sequencing cartridge and any QC processes they follow. The libraries should be ready for sequencing.
SECTION: Bioinformatics
65. Optimal bioinformatics workflows depend on a variety of factors including target taxa, study system, the genetic marker used, computational resources available and various other factors. Other resources with code integration will be much better suited to detailing the steps involved. The process will likely follow a similar progression of steps though:
Read processing: demultiplex, trim primers, merge pairs.
Filtering and error correction: quality filtering, dereplication, denoising, chimera filtering.
Taxonomic unit aggregation: OTU/ASV delimitation.
Taxon assignment: reference database curation, taxon assignment.
SECTION: Ecosystem service provider quantification
66. Following taxon assignment to ASVs, taxa must be assigned as ecosystem service providers, ecosystem disservice providers, or neutral taxa. For an example, see the supplementary information in Cuff et al. (2022).
 

SECTION: Ecosystem service provider quantification
67. Ecosystem service and disservice providers can then be summed for each sample, giving quantities of each which can be analysed against sample metadata.