TITLE: Metabarcoding based gut content analysis of arachnids
AUTHORS: Yuval Zaltz, Evgenia Propistsova, Efrat Gavish-Regev, Jordan P Cuff

[DESCRIPTION]
This protocol describes the workflow used for the gut content analysis via DNA metabarcoding of scorpions and spiders for some work conducted in the Hebrew University of Jerusalem, Israel. The protocol describes sample processing, DNA extraction, PCR amplification, gel electrophoresis, DNA normalisation, PCR-based library preparation, DNA purification and (briefly) sequencing and bioinformatics.

[BEFORE_START]
Plan your experiment carefully. Ensure appropriate safety of all researchers throughout the work. Ensure welfare of the invertebrates used is appropriately considered. For the reduction of sample cross-contamination, consider using separate pre- and post-PCR work spaces and use bleach/Chemgene and/or UV decontamination of surfaces and equipment throughout.

[STEPS]
SECTION: Sample preparation
1. Collect arachnids from study sites, place them into 100 % volume ethanol and store them at -20 °C until ready to proceed with DNA extraction.

 

SECTION: PCR amplification
14. 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).

 

 

 

SECTION: Gel electrophoresis
22. Prepare a gel tank containing sufficient 1X TBE buffer to submerge the gel.

 

SECTION: PCR product normalisation
28. 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 per PCR primer pair 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 25 µL reaction volumes, pipette 20 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: PCR2 and library preparation
50. Quantify the concentration of the library from the previous step (e.g., using Qubit dsDNA assay).

 

SECTION: Sequencing
59. 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. Sequencing times will depend on the machines, protocols and any queues involved at the sequencing facility.
SECTION: Sample preparation
2. Depending on the arachnid, the optimal manner of extracting gut contents can differ. Here, we present two subtly different methods for scorpion and spider gut content analysis. These will be more broadly applicable to other arachnids (e.g., pseudoscorpions, mites, solifugids) but also many other invertebrates.
SECTION: DNA extraction
4. DNA extraction follows the Qiagen DNeasy Blood & Tissue Kit protocol.

Add 180 µL  Buffer ATL to the isolated tissue in the 1.5 mL microcentrifuge tubes.

 

SECTION: DNA extraction
6. Add 20 µL Proteinase K to tubes.

SECTION: DNA extraction
7. Incubate on a thermoshaker at  .

SECTION: DNA extraction
5. Homogenise abdomens with a tissue homogeniser for approximately 3 s  or until fully homogenised.

 

SECTION: DNA extraction
8. Add 200 µL Buffer AL and vortex/shake to mix.

SECTION: DNA extraction
9. Add 200 µL 100 % (v/v) ethanol and vortex/shake to mix

SECTION: DNA extraction
10. Pipette the mixture (~600 µL) into a spin column held within a 2 mL collection tube and centrifuge for 6000 x g, 1 min. Discard the flow-through and collection tube.

 

SECTION: DNA extraction
11. Place the spin column into a new collection tube, add 500 µL Buffer AW1 and centrifuge for 6000 x g, 1 min. Discard the flow-through and collection tube.

SECTION: DNA extraction
12. Place the spin column into a new collection tube, add 500 µL Buffer AW2 and centrifuge for 20000 x g, 3 min. Discard the flow-through and collection tube.

SECTION: DNA extraction
13. Place the spin column into a new 1.5 mL microcentrifuge tube, add 200 µL Buffer AE, incubate at 25 Room temperature for 1 min and centrifuge for 6000 x g, 1 min.


 

3. Using bleach-sterilised tools, cut the scorpion's abdomen open and scrape the innards of the abdomen into a 1.5 mL microcentrifuge tube.

 

 

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

 

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

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

   AB1ReagentVolume (μL)2Molecular grade water (DNase free)422.432X hot-start PCR mastermix528

 

SECTION: PCR amplification
17. For ease, if able to use a 96-well pipette, consider creating a "primer plate" containing both PCR primers for each well at 2.5 micromolar (µM) concentration; this is especially effective when using multiple plates. Add 20 µL mastermix to each well of the PCR plate, followed by 2 µL of each primer mix from the primer plate to its corresponding well in the PCR plate. It is possible to use this strategy with a multi-channel or even single-channel pipette, but the alternative described immediately below may be easier. 

If not using a 96-well pipette, consider making eight mastermixes each with sufficient reagents for 12 samples (20 µL µL mastermix per well, so 264 µL µL for 12 with some overage for pipetting error), each containing a different forward primer (with 2 µL µL of 2.5 micromolar (µM) forward primer per well, so 26.4 µL µL per mastermix for 12 with some overage for pipetting error). Distribute 22 µL µL of forward primer + mastermix to each well across the rows corresponding to each forward primer. Then distribute 2 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 amplification
18. 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 should be added as samples).

SECTION: PCR amplification
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 amplification
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 amplification
21. 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 concentrations. Gel electrophoresis is nevertheless described below.
SECTION: PCR product normalisation
29. 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: PCR product normalisation
29.1. If using beads such as Sera-Mag Magnetic SpeedBeads (carboxylated,  , 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 % volumeTween 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: PCR product normalisation
29.2. To the beads, add the following mix:

   AB1ReagentVolume25 M NaCl25 mL3Molecular grade water3.582 mL41 N HCl0.168 mL51 M Tris base0.5 mL60.1 M disodium EDTA0.5 mL

SECTION: PCR product normalisation
29.3. 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: PCR product normalisation
29.4. Add 5 mL of 1X bead solution to 45 mL of the following mix to make a 0.1X solution:

   AB1ReagentVolume25 M NaCl25 mL3Molecular grade water3.582 mL41 N HCl0.168 mL51 M Tris base0.5 mL60.1 M disodium EDTA0.5 mL750 % PEG20 mL

SECTION: PCR product normalisation
30. 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 PCR product is available, add a volume equivalent to the available PCR product volume of 0.1X SPRI beads to a new 96-well plate.

 

SECTION: PCR product normalisation
31. 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: PCR product normalisation
32. Incubate at Room temperature for 5 min .

SECTION: PCR product normalisation
33. Place on a magnetic stand for 5 min.

SECTION: PCR product normalisation
34. 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).

SECTION: PCR product normalisation
35. Add 200 µL 80 % volume ethanol to each well.

SECTION: PCR product normalisation
36. Remove the ethanol and add a further 200 µL 80 % volume ethanol to each well.

SECTION: PCR product normalisation
37. Remove the ethanol as completely as possible by pipetting.

 

SECTION: PCR product normalisation
38. Add 10 µL molecular grade water to each well, shake at   and incubate at Room temperature for 5 min.

SECTION: PCR product normalisation
39. Place on a magnetic stand for 5 min.

SECTION: PCR product normalisation
40. Pool 8 µL from each well into a single tube, leaving the magnetic beads behind in the 96-well plate.

 

SECTION: PCR product normalisation
41. Mix the pooled samples by flicking and inverting, and transfer 700 µL into a new 1.5 mL microcentrifuge tube.

SECTION: PCR product normalisation
42. Add 700 µL 1X magnetic beads for DNA purification (see note below for size selection) and mix by vortexing. Incubate at Room temperature for 5 min.

 

SECTION: PCR product normalisation
43. Place on a magnetic stand for 5 min.

SECTION: PCR product normalisation
44. Add 400 µL 80 % volume ethanol.

SECTION: PCR product normalisation
45. Remove the ethanol and add a further 400 µL 80 % volume ethanol.

SECTION: PCR product normalisation
46. 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: PCR product normalisation
47. Add 100 µL molecular grade water and mix by vortexing. Incubate at Room temperature for 5 min.

SECTION: PCR product normalisation
48. Place the sample on a magnetic stand for 5 min.

SECTION: PCR product normalisation
49. Remove 95 µL of the water and place it in a new tube. This will be the library used in the subsequent section.

SECTION: PCR2 and library preparation
51. Dilute library to ≤5000 µg/µL (≤5 ng/µL) in molecular grade water.

 

SECTION: PCR2 and library preparation
52. Add a drop of mineral oil to one PCR tube for each library, alongside an additional tube for a negative control.
SECTION: PCR2 and library preparation
53. Per library, assemble the following reaction:

   AB1ReagentVolume22X hot-start PCR mastermix7.53Molecular grade water14Nextera sequencing adapter primer mix1.55DNA library5

 
SECTION: PCR2 and library preparation
54. Briefly centrifuge the tubes to ensure that the oil is above the PCR mix.
SECTION: PCR2 and library preparation
55. 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: PCR2 and library preparation
56. Quantify the concentration of the library from the previous step (e.g., using Qubit dsDNA assay).
SECTION: PCR2 and library preparation
57. 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: PCR2 and library preparation
58. 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: Bioinformatics
60. 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: Sample preparation
3. Using bleach-sterilised tools, remove the spider's abdomen and place it into a 1.5 mL microcentrifuge tube.

 

 

SECTION: Gel electrophoresis
23. Prepare a 2 Mass / % volume agarose gel for the samples to go into (detailed in the below substeps).

SECTION: Gel electrophoresis
23.2. Determine a suitable size (and therefore volume) of gel based on the size of the tank available. For a 150 mL gel, weigh 3 g of agarose and add it to 150 mL of 1X TBE buffer. 

SECTION: Gel electrophoresis
23.3. Heat this mixture in a microwave in a conical flask until it begins bubbling.
SECTION: Gel electrophoresis
23.4. Mix the solution by gently swirling it and heat again until bubbling. Repeat this as necessary until there are no large lumps of agarose remaining.

 

SECTION: Gel electrophoresis
23.5. Add 3 µL of gel dye (e.g., SYBRSafe) and mix by swirling.

SECTION: Gel electrophoresis
23.1. Prepare a gel mould with sufficient comb tips for the number of samples to be run. Ensure these are spaced out enough to allow the gel to run and separate without running into another well.
SECTION: Gel electrophoresis
23.6. Pour the mixture into the gel mould and allow to set for 25 min.

SECTION: Gel electrophoresis
24. Add 2 µL of each sample to a new plate and add 2 µL of 1X DNA dye for visualisation of the products in the gel.

SECTION: Gel electrophoresis
25. Once set, place the gel into the tank and add 4 µL of DNA gel ladder to each side of the gel in each row, and then 4 µL of dyed PCR product to each other well. 

 


SECTION: Gel electrophoresis
26. Once filled, run the gel power pack at 120 V and 120 A for 25 min.

SECTION: Gel electrophoresis
27. Visualise the gel under a UV or blue light in a gel dock to confirm amplification and a lack of contamination in negative controls.

 

SECTION: DNA extraction
4. DNA extraction follows the Qiagen DNeasy Blood & Tissue Kit protocol.

Add 180 µL  Buffer ATL to the isolated tissue in the 1.5 mL microcentrifuge tubes.

 

SECTION: DNA extraction
5. Homogenise abdomens with a tissue homogeniser for approximately 3 s  or until fully homogenised.

 

SECTION: DNA extraction
6. Add 20 µL Proteinase K to tubes.

SECTION: DNA extraction
7. Incubate on a thermoshaker at  .

SECTION: DNA extraction
8. Add 200 µL Buffer AL and vortex/shake to mix.

SECTION: DNA extraction
9. Add 200 µL 100 % (v/v) ethanol and vortex/shake to mix

SECTION: DNA extraction
10. Pipette the mixture (~600 µL) into a spin column held within a 2 mL collection tube and centrifuge for 6000 x g, 1 min. Discard the flow-through and collection tube.

 

SECTION: DNA extraction
11. Place the spin column into a new collection tube, add 500 µL Buffer AW1 and centrifuge for 6000 x g, 1 min. Discard the flow-through and collection tube.

SECTION: DNA extraction
12. Place the spin column into a new collection tube, add 500 µL Buffer AW2 and centrifuge for 20000 x g, 3 min. Discard the flow-through and collection tube.

SECTION: DNA extraction
13. Place the spin column into a new 1.5 mL microcentrifuge tube, add 200 µL Buffer AE, incubate at 25 Room temperature for 1 min and centrifuge for 6000 x g, 1 min.


 

SECTION: PCR amplification
14. 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).

 

 

 

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

 

SECTION: PCR amplification
17. For ease, if able to use a 96-well pipette, consider creating a "primer plate" containing both PCR primers for each well at 2.5 micromolar (µM) concentration; this is especially effective when using multiple plates. Add 20 µL mastermix to each well of the PCR plate, followed by 2 µL of each primer mix from the primer plate to its corresponding well in the PCR plate. It is possible to use this strategy with a multi-channel or even single-channel pipette, but the alternative described immediately below may be easier. 

If not using a 96-well pipette, consider making eight mastermixes each with sufficient reagents for 12 samples (20 µL µL mastermix per well, so 264 µL µL for 12 with some overage for pipetting error), each containing a different forward primer (with 2 µL µL of 2.5 micromolar (µM) forward primer per well, so 26.4 µL µL per mastermix for 12 with some overage for pipetting error). Distribute 22 µL µL of forward primer + mastermix to each well across the rows corresponding to each forward primer. Then distribute 2 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 amplification
16. Prepare enough PCR mastermix for each sample.

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

   AB1ReagentVolume (μL)2Molecular grade water (DNase free)422.432X hot-start PCR mastermix528

 

SECTION: PCR amplification
18. 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 should be added as samples).

SECTION: PCR amplification
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 amplification
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 amplification
21. 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 concentrations. Gel electrophoresis is nevertheless described below.
SECTION: Gel electrophoresis
22. Prepare a gel tank containing sufficient 1X TBE buffer to submerge the gel.

 

SECTION: Gel electrophoresis
23. Prepare a 2 Mass / % volume agarose gel for the samples to go into (detailed in the below substeps).

SECTION: Gel electrophoresis
23.1. Prepare a gel mould with sufficient comb tips for the number of samples to be run. Ensure these are spaced out enough to allow the gel to run and separate without running into another well.
SECTION: Gel electrophoresis
23.2. Determine a suitable size (and therefore volume) of gel based on the size of the tank available. For a 150 mL gel, weigh 3 g of agarose and add it to 150 mL of 1X TBE buffer. 

SECTION: Gel electrophoresis
23.3. Heat this mixture in a microwave in a conical flask until it begins bubbling.
SECTION: Gel electrophoresis
23.4. Mix the solution by gently swirling it and heat again until bubbling. Repeat this as necessary until there are no large lumps of agarose remaining.

 

SECTION: Gel electrophoresis
23.5. Add 3 µL of gel dye (e.g., SYBRSafe) and mix by swirling.

SECTION: Gel electrophoresis
23.6. Pour the mixture into the gel mould and allow to set for 25 min.

SECTION: Gel electrophoresis
24. Add 2 µL of each sample to a new plate and add 2 µL of 1X DNA dye for visualisation of the products in the gel.

SECTION: Gel electrophoresis
25. Once set, place the gel into the tank and add 4 µL of DNA gel ladder to each side of the gel in each row, and then 4 µL of dyed PCR product to each other well. 

 


SECTION: Gel electrophoresis
26. Once filled, run the gel power pack at 120 V and 120 A for 25 min.

SECTION: Gel electrophoresis
27. Visualise the gel under a UV or blue light in a gel dock to confirm amplification and a lack of contamination in negative controls.

 

SECTION: PCR product normalisation
28. 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 per PCR primer pair 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 25 µL reaction volumes, pipette 20 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: PCR product normalisation
29. 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: PCR product normalisation
29.1. If using beads such as Sera-Mag Magnetic SpeedBeads (carboxylated,  , 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 % volumeTween 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: PCR product normalisation
29.2. To the beads, add the following mix:

   AB1ReagentVolume25 M NaCl25 mL3Molecular grade water3.582 mL41 N HCl0.168 mL51 M Tris base0.5 mL60.1 M disodium EDTA0.5 mL

SECTION: PCR product normalisation
29.3. 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: PCR product normalisation
29.4. Add 5 mL of 1X bead solution to 45 mL of the following mix to make a 0.1X solution:

   AB1ReagentVolume25 M NaCl25 mL3Molecular grade water3.582 mL41 N HCl0.168 mL51 M Tris base0.5 mL60.1 M disodium EDTA0.5 mL750 % PEG20 mL

SECTION: PCR product normalisation
30. 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 PCR product is available, add a volume equivalent to the available PCR product volume of 0.1X SPRI beads to a new 96-well plate.

 

SECTION: PCR product normalisation
31. 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: PCR product normalisation
32. Incubate at Room temperature for 5 min .

SECTION: PCR product normalisation
33. Place on a magnetic stand for 5 min.

SECTION: PCR product normalisation
34. 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).

SECTION: PCR product normalisation
35. Add 200 µL 80 % volume ethanol to each well.

SECTION: PCR product normalisation
36. Remove the ethanol and add a further 200 µL 80 % volume ethanol to each well.

SECTION: PCR product normalisation
37. Remove the ethanol as completely as possible by pipetting.

 

SECTION: PCR product normalisation
38. Add 10 µL molecular grade water to each well, shake at   and incubate at Room temperature for 5 min.

SECTION: PCR product normalisation
39. Place on a magnetic stand for 5 min.

SECTION: PCR product normalisation
40. Pool 8 µL from each well into a single tube, leaving the magnetic beads behind in the 96-well plate.

 

SECTION: PCR product normalisation
41. Mix the pooled samples by flicking and inverting, and transfer 700 µL into a new 1.5 mL microcentrifuge tube.

SECTION: PCR product normalisation
42. Add 700 µL 1X magnetic beads for DNA purification (see note below for size selection) and mix by vortexing. Incubate at Room temperature for 5 min.

 

SECTION: PCR product normalisation
43. Place on a magnetic stand for 5 min.

SECTION: PCR product normalisation
44. Add 400 µL 80 % volume ethanol.

SECTION: PCR product normalisation
45. Remove the ethanol and add a further 400 µL 80 % volume ethanol.

SECTION: PCR product normalisation
46. 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: PCR product normalisation
47. Add 100 µL molecular grade water and mix by vortexing. Incubate at Room temperature for 5 min.

SECTION: PCR product normalisation
48. Place the sample on a magnetic stand for 5 min.

SECTION: PCR product normalisation
49. Remove 95 µL of the water and place it in a new tube. This will be the library used in the subsequent section.

SECTION: PCR2 and library preparation
50. Quantify the concentration of the library from the previous step (e.g., using Qubit dsDNA assay).

 

SECTION: PCR2 and library preparation
51. Dilute library to ≤5000 µg/µL (≤5 ng/µL) in molecular grade water.

 

SECTION: PCR2 and library preparation
52. Add a drop of mineral oil to one PCR tube for each library, alongside an additional tube for a negative control.
SECTION: PCR2 and library preparation
53. Per library, assemble the following reaction:

   AB1ReagentVolume22X hot-start PCR mastermix7.53Molecular grade water14Nextera sequencing adapter primer mix1.55DNA library5

 
SECTION: PCR2 and library preparation
54. Briefly centrifuge the tubes to ensure that the oil is above the PCR mix.
SECTION: PCR2 and library preparation
55. 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: PCR2 and library preparation
56. Quantify the concentration of the library from the previous step (e.g., using Qubit dsDNA assay).
SECTION: PCR2 and library preparation
57. 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: PCR2 and library preparation
58. 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: Sequencing
59. 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. Sequencing times will depend on the machines, protocols and any queues involved at the sequencing facility.
SECTION: Bioinformatics
60. 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.