TITLE: High-throughput individual insect metabarcoding for identification and interaction data
AUTHORS: Jordan P Cuff, Thomas Howells, James JN Kitson, Ben SJ Hawthorne, darren.evans

[DESCRIPTION]
This protocol is designed for extracting DNA from individual invertebrates for metabarcoding to detect and identify ecosystem service and disservice providers and their interactions. The reagents and methods proposed offer a cost effective and high-throughput method for molecular analyses of individual invertebrates using standard lab equipment. Where specialist equipment us used, attempts are made to suggest low-cost alternatives.

[STEPS]
SECTION: Sampling of invertebrates
1. Select suitable sites and locations for insect sampling. This protocol is based on hand-collected foliar and flower visitor invertebrates, but other collection methods are viable; be wary of cross-contamination and surface DNA though, and consider surface sterilisation accordingly. Consider how systematic the study needs to be and the various constraints imposed on the data by the study design.
SECTION: DNA extraction: purification
1. Add 200 μL of the supernatant from STEP 10 into each well containing Protein Denaturation Buffer and ethanol. 
SECTION: DNA extraction: purification
0.1. Per 96-well plate, the Protein Denaturation Buffer and ethanol master mix should comprise:

 

SECTION: DNA extraction: purification
2. 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
3. Centrifuge at ≥ 6000 x g for 1 minute and discard the flow-through.
 

SECTION: DNA extraction: purification
4. Add 500 μL Wash Buffer 1 to each spin column and cover with a breathable seal.
SECTION: DNA extraction: purification
5. Centrifuge at ≥ 6000 x g for 1 minute and discard the flow-through.
 

SECTION: DNA extraction: purification
4.1. Wash Buffer 1 should be comprised of the following reagents:

 

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

 

SECTION: DNA extraction: purification
7. Centrifuge at 20,000 x g for 3 minutes and discard the flow-through.
 

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

SECTION: DNA extraction: purification
9. 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
9.1. Elution Buffer should be pH ~7 and comprised of the following reagents:
 
 

SECTION: DNA extraction: purification
10. 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
10. 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
10.1. Protein Denaturation Buffer should be comprised of the following reagents:
  
 

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

 

SECTION: DNA extraction: purification
11. 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
12. Centrifuge at ≥ 6000 x g for 10 minutes and discard the flow-through.
 

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

 

SECTION: DNA extraction: purification
14. Centrifuge at ≥ 6000 x g for 5 minutes and discard the flow-through.
 

SECTION: DNA extraction: purification
15. Add 500 μL Wash Buffer 2 to each spin column and cover with a breathable seal.
SECTION: DNA extraction: purification
17. Carefully move the spin column plate to a new 0.5 mL DNA collection plate.
 

SECTION: DNA extraction: purification
15.1. Wash Buffer 2 should be pH ~7 and comprised of the following reagents:
 
 

SECTION: DNA extraction: purification
16. Centrifuge at ≥ 6000 x g for 15 minutes and discard the flow-through.
 

SECTION: DNA extraction: purification
18. 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
18.1. Elution Buffer should be pH ~7 and comprised of the following reagents:
 
 

SECTION: DNA extraction: purification
19. 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
1. Grind the larvae in a Geno/Grinder 2010 at full speed (1750 RPM) for 4 minutes.
 

SECTION: Initial digestion of OPM larvae
2. Centrifuge at 4,000 g for 2 minutes.
SECTION: Initial digestion of OPM larvae
3. Remove the ball bearings using a magnet, taking care not to cross-contaminate the samples.
SECTION: Initial digestion of OPM larvae
4. 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
4.1. Lysis Buffer 2 should be pH 9 and comprised of the following reagents:
 
 

SECTION: Initial digestion of OPM larvae
4.2. Per sample, the Proteinase Buffer master mix should comprise:

 

SECTION: Initial digestion of OPM larvae
5. Incubate at 37 °C overnight (12-16 hours)
 

SECTION: Initial digestion of OPM larvae
6. Centrifuge at 4000 x g for 4 minutes.
SECTION: Initial digestion of OPM larvae
7. 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
8. Centrifuge at 2000 x g for 4 minutes.
SECTION: Initial digestion of OPM larvae
9. Transfer 200 μL of the supernatant to a clean 2.2 mL deep-well 96-well plate. 
 

SECTION: Preparation and homogenisation of samples
7. Add one 3 mm hardened carbon steel bead to each well.
 

SECTION: Sampling of invertebrates
2. Following collection, store insects in sterile tubes. For flower visitors, store insects dry and consider killing them with ethyl acetate on a cotton ball; for other insects, storage in 100 % ethanol may be ideal. Kill invertebrates as humanely as possible. Freezing is widely considered to be among the optimal methods.
SECTION: Sampling of invertebrates
3. Transfer individual invertebrates into 96-well plates (ideally deep-well, e.g., 2.2 mL, with fixable lids for grinding/washing). Consider the distribution of experimental controls ahead of subsequent steps to streamline downstream liquid handling.

 

Store samples at -20 °C until ready to process.
SECTION: DNA extraction
11. Centrifuge the plate at 2000 x g, 2 min.


SECTION: Preparation and homogenisation of samples
5. Add 100 µL  TNES lysis buffer to each well of a 96-well plate.

 

SECTION: Preparation and homogenisation of samples
6. Two protocols are presented below, which simply differ in whether samples are ground or surface-washed. For herbivorous insects, we recommend grinding to facilitate analysis of internal DNA (either consumed plants or parasitoids). For flower visitors, we recommend surface washing (exactly the same protocol, but without ball bearings during the grinding step). This still facilitates analysis of DNA from the insect itself, but also pollen on the surface of the insect. These protocols could both be used for a wider range of potential applications.
SECTION: Preparation and homogenisation of samples
8. Grind the samples in a tissue grinder/homogeniser/lyser at 1750 RPM for 1 minute.
SECTION: DNA extraction
12. Prepare Kingfisher Apex reagent plates as below.

 

SECTION: Preparation and homogenisation of samples
9. Add 10 µL 20 mg/mL papain to each well.

 

SECTION: Preparation and homogenisation of samples
10. Incubate overnight (~960 min) at 37 °C .

SECTION: DNA extraction
13. Insert plates into the Kingfisher Apex and run a preset programme with the below steps.
SECTION: PCR
17. 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. For 10 µL reaction volumes, this will subsequently involve adding 7.75 µL  hot-start Taq polymerase and water mastermix (described in the step above) 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 micromolar (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: PCR
18. 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: PCR
20. 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: PCR
21. 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: Normalisation
24. 
 

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: Normalisation
25. 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: Normalisation
27. 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 ( ).

 

SECTION: Normalisation
29. Place on a magnetic stand for 5 min.
SECTION: Library preparation and sequencing
46. Quantify the concentration of the library from the previous step (e.g., using Qubit dsDNA assay).
SECTION: Library preparation and sequencing
47. Dilute library to ≤ 5 ng/µL in molecular grade water.

 

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

 

SECTION: Library preparation and sequencing
50. Briefly centrifuge the tubes to ensure that the oil is above the PCR mix.
SECTION: Library preparation and sequencing
51. 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. 
SECTION: Library preparation and sequencing
53. Quantify the concentration of the library from the previous step (e.g., using Qubit dsDNA assay).
SECTION: Library preparation and sequencing
54. 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 extraction
12.1. For the sample plate, to each well of a 96-well deep-well plate, add 60 µL of the lysate from STEP 11, 120 µL of 1.5X GITC buffer, 120 µL of 1 mg/mL SeraMag Speed Beads in TE (10 millimolar (mM) Tris and 1 millimolar (mM) EDTA) and 240 µL isopropanol.

 

SECTION: DNA extraction
12.2. For the two ethanol plates, to each well of two 96-well deep-well plates, add 400 µL 80 % ethanol.

SECTION: DNA extraction
12.3. For the isopropanol plate, to each well of a 96-well deep-well plate, add 400 µL isopropanol.

SECTION: DNA extraction
12.4. For the elution plate, to each well of a 96-well standard plate, add 100 µL molecular biology grade water.

SECTION: DNA extraction
13.1. Pick up the 96 deep-well tip comb from a 96-well standard plate.
SECTION: DNA extraction
13.2. Bind DNA to the beads by mixing at medium speed for 5 min with a slow post-mix for 5 min.

SECTION: DNA extraction
13.3. Collect the beads in three 1 s collections.

SECTION: DNA extraction
13.4. Release the beads into the isopropanol plate and mix for 1 min at medium speed, and collect the beads in three 1 s collections.


SECTION: DNA extraction
13.5. Release the beads into one of the 80 % ethanol plates and mix for 1 min at medium speed, and collect the beads in three 1 s collections.
SECTION: DNA extraction
13.6. Release the beads into one of the 80 % ethanol plates and mix for 1 min at medium speed, and collect the beads in three 1 s collections.
SECTION: DNA extraction
13.7. Dry the beads above the well for 2 min.

SECTION: DNA extraction
13.8. Release the beads into the elution plate and mix for 2 min at fast speed with a slow post-mix for 3 min, and collect the beads in four 20 s collections.
SECTION: DNA extraction
13.9. Leave the tip comb in an empty 96-well standard plate.
SECTION: DNA extraction
14. Store the eluted DNA at -20 °C until ready for subsequent steps.

SECTION: PCR
15. 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: PCR
16. Prepare enough PCR mastermix for each sample.

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

 

 

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

 


SECTION: PCR
23. 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: Normalisation
25.1. 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 millimolar (mM) Tris base, 1 millimolar (mM) EDTA, 0.05 % volume 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: Normalisation
25.2. To the beads, add the following mix:

 

SECTION: Normalisation
25.3. Add 20 mL of 50 % volume PEG to the tube to reach a 1X bead solution (alongside making the 0.1X solution, this will be useful later).

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

 

SECTION: Normalisation
26. 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: Normalisation
28. Incubate at Room temperature for 5 min.

SECTION: Normalisation
30. 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 solutions).

SECTION: Normalisation
31. Add 200 µL 80 % volume ethanol to each well.

SECTION: Normalisation
32. Remove the ethanol and add a further 200 µL 80 % volume ethanol to each well.
SECTION: Normalisation
33. Remove the ethanol as completely as possible by pipetting.

 

SECTION: Normalisation
34. Add 10 µL molecular grade water to each well, shake at   and incubate at Room temperature  for 5 min.

SECTION: Normalisation
35. Place on a magnetic stand for 5 min.

SECTION: Normalisation
36. Pool 8 µL from each well into a single tube, leaving magnetic beads behind in the 96-well plate.

 

SECTION: Normalisation
37. Mix the pooled samples by flicking and inverting, and transfer 700 µL into a new 2.5 mL microcentrifuge tube.

SECTION: Normalisation
38. Add 700 µL 1X magnetic beads for DNA purification (see note below for size selection) and mix by vortexing. Incubate for 5 min.

 

SECTION: Normalisation
39. Place on a magnetic stand for 5 min.

SECTION: Normalisation
40. Add 400 µL 80 % volume ethanol.

SECTION: Normalisation
41. Remove the ethanol and add a further 400 µL 80 % volume ethanol.

SECTION: Normalisation
42. 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: Normalisation
43. Add 100 µL molecular grade water and mix by vortexing. Incubate for 5 min at Room temperature .

SECTION: Normalisation
44. Place the sample on a magnetic stand for 5 min.

SECTION: Normalisation
45. Remove 95 µL of the water and place it in a new tube. This will be the library used in the subsequent section.

SECTION: Library preparation and sequencing
55. 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: Library preparation and sequencing
56. 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: PCR
22. Run your PCR programme.
SECTION: Library preparation and sequencing
52. Run your PCR programme for 12-20 cycles, with 12 for ~5 ng/µL libraries and more for lower concentrations.

SECTION: Preparation and homogenisation of samples
4. The DNA extraction protocol is largely adapted from the BOMB-Bio tissue nucleic acid extraction protocol. See their documentation for additional detail.

 

SECTION: Preparation and homogenisation of samples
7. Wash pollen and surface DNA (including DNA of the insect itself) from the samples in a tissue grinder/homogeniser/lyser at 1750 RPM for 1 minute.


SECTION: Preparation and homogenisation of samples
8. Add 10 µL 20 mg/mL papain to each well.

 

SECTION: Preparation and homogenisation of samples
9. Incubate overnight (~960 min) at 37 °C .

SECTION: PCR
14. 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: PCR
15. Prepare enough PCR mastermix for each sample.

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

 

 

SECTION: PCR
16. 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. For 10 µL reaction volumes, this will subsequently involve adding 7.75 µL  hot-start Taq polymerase and water mastermix (described in the step above) 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 micromolar (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: PCR
17. 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: PCR
18. Distribute one drop of mineral oil into each well of the PCR plate(s) (~20 µL). 

 


SECTION: PCR
19. 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: PCR
20. 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: PCR
21. Run your PCR programme.
SECTION: PCR
22. 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: Normalisation
23. 
 

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: Normalisation
24. 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: Normalisation
24.1. 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 millimolar (mM) Tris base, 1 millimolar (mM) EDTA, 0.05 % volume 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: Normalisation
24.2. To the beads, add the following mix:

 

SECTION: Normalisation
24.3. Add 20 mL of 50 % volume PEG to the tube to reach a 1X bead solution (alongside making the 0.1X solution, this will be useful later).

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

 

SECTION: Normalisation
25. 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: Normalisation
26. 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 ( ).

 

SECTION: Normalisation
27. Incubate at Room temperature for 5 min.

SECTION: Normalisation
28. Place on a magnetic stand for 5 min.
SECTION: Normalisation
29. 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 solutions).

SECTION: Normalisation
30. Add 200 µL 80 % volume ethanol to each well.

SECTION: Normalisation
31. Remove the ethanol and add a further 200 µL 80 % volume ethanol to each well.
SECTION: Normalisation
32. Remove the ethanol as completely as possible by pipetting.

 

SECTION: Normalisation
33. Add 10 µL molecular grade water to each well, shake at   and incubate at Room temperature  for 5 min.

SECTION: Normalisation
34. Place on a magnetic stand for 5 min.

SECTION: Normalisation
35. Pool 8 µL from each well into a single tube, leaving magnetic beads behind in the 96-well plate.

 

SECTION: Normalisation
36. Mix the pooled samples by flicking and inverting, and transfer 700 µL into a new 2.5 mL microcentrifuge tube.

SECTION: Normalisation
37. Add 700 µL 1X magnetic beads for DNA purification (see note below for size selection) and mix by vortexing. Incubate for 5 min.

 

SECTION: Normalisation
38. Place on a magnetic stand for 5 min.

SECTION: Normalisation
39. Add 400 µL 80 % volume ethanol.

SECTION: Normalisation
40. Remove the ethanol and add a further 400 µL 80 % volume ethanol.

SECTION: Normalisation
41. 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: Normalisation
42. Add 100 µL molecular grade water and mix by vortexing. Incubate for 5 min at Room temperature .

SECTION: Normalisation
43. Place the sample on a magnetic stand for 5 min.

SECTION: Normalisation
44. Remove 95 µL of the water and place it in a new tube. This will be the library used in the subsequent section.

SECTION: Library preparation and sequencing
45. Quantify the concentration of the library from the previous step (e.g., using Qubit dsDNA assay).
SECTION: Library preparation and sequencing
46. Dilute library to ≤ 5 ng/µL in molecular grade water.

 

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

 

SECTION: Library preparation and sequencing
49. Briefly centrifuge the tubes to ensure that the oil is above the PCR mix.
SECTION: Library preparation and sequencing
50. 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. 
SECTION: Library preparation and sequencing
51. Run your PCR programme for 12-20 cycles, with 12 for ~5 ng/µL libraries and more for lower concentrations.

SECTION: Library preparation and sequencing
52. Quantify the concentration of the library from the previous step (e.g., using Qubit dsDNA assay).
SECTION: Library preparation and sequencing
53. 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: Library preparation and sequencing
54. 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: Library preparation and sequencing
55. 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 extraction
10. Centrifuge the plate at 2000 x g, 2 min.

SECTION: DNA extraction
11. Prepare Kingfisher Apex reagent plates as below.

 

SECTION: DNA extraction
11.1. For the sample plate, to each well of a 96-well deep-well plate, add 60 µL of the lysate from STEP 10, 120 µL of 1.5X GITC buffer, 120 µL of 1 mg/mL SeraMag Speed Beads in TE (10 millimolar (mM) Tris and 1 millimolar (mM) EDTA) and 240 µL isopropanol.

 

SECTION: DNA extraction
11.2. For the two ethanol plates, to each well of two 96-well deep-well plates, add 400 µL 80 % ethanol.

SECTION: DNA extraction
11.3. For the isopropanol plate, to each well of a 96-well deep-well plate, add 400 µL isopropanol.

SECTION: DNA extraction
11.4. For the elution plate, to each well of a 96-well standard plate, add 100 µL molecular biology grade water.

SECTION: DNA extraction
12. Insert plates into the Kingfisher Apex and run a preset programme with the below steps.
SECTION: DNA extraction
12.1. Pick up the 96 deep-well tip comb from a 96-well standard plate.
SECTION: DNA extraction
12.2. Bind DNA to the beads by mixing at medium speed for 5 min with a slow post-mix for 5 min.

SECTION: DNA extraction
12.3. Collect the beads in three 1 s collections.

SECTION: DNA extraction
12.4. Release the beads into the isopropanol plate and mix for 1 min at medium speed, and collect the beads in three 1 s collections.


SECTION: DNA extraction
12.5. Release the beads into one of the 80 % ethanol plates and mix for 1 min at medium speed, and collect the beads in three 1 s collections.
SECTION: DNA extraction
12.6. Release the beads into one of the 80 % ethanol plates and mix for 1 min at medium speed, and collect the beads in three 1 s collections.
SECTION: DNA extraction
12.7. Dry the beads above the well for 2 min.

SECTION: DNA extraction
12.8. Release the beads into the elution plate and mix for 2 min at fast speed with a slow post-mix for 3 min, and collect the beads in four 20 s collections.
SECTION: DNA extraction
12.9. Leave the tip comb in an empty 96-well standard plate.
SECTION: DNA extraction
13. Store the eluted DNA at -20 °C until ready for subsequent steps.