TITLE: Biofilm DNA metabarcoding protocol
AUTHORS: Amy Thorpe, Tim Goodall, Lindsay Kate Newbold, Joe Taylor, Jonathan Warren, kerry.walsh, Daniel S Read

[DESCRIPTION]
Full protocol for the extraction of DNA from river biofilm samples, 2-step PCR amplification of 16S rRNA, 18S rRNA, ITS2 and rbcL gene regions and amplicon sequencing.

[STEPS]
SECTION: 1. DNA extraction
1. Defrost and gently vortex samples to resuspend sample material.
 

SECTION: 1. DNA extraction
2. Transfer 100 µL of sample material to each tube of the 96-tube lysis rack. Make a note of sample positions and leave at least one tube free of sample material as a negative extraction control.
SECTION: 1. DNA extraction
3. Add 500 µL of Zymo DNA/RNA shield to each tube of the lysis rack and seal rack with provided tube cap films.
SECTION: 1. DNA extraction
4. Secure in a bead beater such as the Qiagen TissueLyser II and lyse at 20 Hz for 20 min.
SECTION: 1. DNA extraction
5. Before uncapping the tube rack, centrifuge at 3000 x g for 5 min.
SECTION: 1. DNA extraction
6. Add 20 µL of Proteinase K to each tube of the lysis rack.
SECTION: 1. DNA extraction
7. Incubate at 65 °C for 20 min.
SECTION: 1. DNA extraction
10. Add 750 µL beta-mercaptoethanol (BME) to the genomic lysis buffer and invert the bottle to mix. 
 

SECTION: 1. DNA extraction
9. Transfer 250 µL of lysed supernatant from the lysis rack to the same position in a 96-well block.
SECTION: 1. DNA extraction
8. Before uncapping the tube rack, centrifuge at 3000 x g for 5 min.
 

SECTION: 1. DNA extraction
12. Centrifuge at 3000 x g for 5 min.
SECTION: 1. DNA extraction
11. Add 750 µL of genomic lysis buffer with BME added to each well of the 96-well block, pipette up and down to mix. Seal with an adhesive plate seal.
SECTION: 1. DNA extraction
13. Remove the plate seal and transfer 500 µL from each well of the 96-well block to the same position in a silicon-A plate mounted on a collection plate. Seal the silicon-A plate with an adhesive plate seal.
SECTION: 1. DNA extraction
14. Centrifuge at 3000 x g for 5 min. Discard the flow through from the collection plate into a hazardous waste bottle and return the silicon-A plate to the same collection plate.   until all supernatant has been processed.
SECTION: 1. DNA extraction
15. Remove the plate seal and add 200 µL of DNA pre-wash buffer to each well of the silicon-A plate mounted on the emptied collection plate. Replace the adhesive plate seal.
SECTION: 1. DNA extraction
16. Centrifuge at 3000 x g for 5 min. Discard the flow through from the collection plate into a hazardous waste bottle and return the silicon-A plate to the same collection plate.
SECTION: 1. DNA extraction
17. Remove the plate seal and add 500 µL of g-DNA wash buffer to each well of the silicon-A plate mounted on the emptied collection plate. Replace the adhesive plate seal.
SECTION: 1. DNA extraction
18. Centrifuge at 3000 x g for 5 min. Discard the flow through from the collection plate into a hazardous waste bottle.
 

SECTION: 1. DNA extraction
19. Place a HRC plate on an elution plate and pierce the cover foil. Add 150 µL of prep solution to each well of the HRC plate and leave at room temperature for 5 min.
SECTION: 1. DNA extraction
20. Centrifuge at 3000 x g for 5 min. Discard the flow through from the elution plate into a hazardous waste bottle.
SECTION: 1. DNA extraction
21. Remove the plate seal from the silicon-A plate and place onto the prepared HRC plate, place the assembly onto a new elution plate to form a 3-plate stack. Add 100 µL of DNA elution buffer to the silicon-A plate and replace the adhesive plate seal.
SECTION: 1. DNA extraction
22. Centrifuge at 3500 x g for 5 min.
SECTION: 1. DNA extraction
23. Seal the elution plate with an adhesive plate seal, label and store at 4 °C in a fridge short-term (up to 1 week) or in a freezer at -20 °C to -70 °C for long term storage.
SECTION: 2. DNA quantification
24. To quantify using Nanodrop, clean each pedestal of the Nanodrop spectrophotometer with molecular grade water and blank the instrument with 1 µL of elution buffer as instructed by the Nanodrop software. Transfer 1 µL of each DNA sample to a pedestal, shut the lid and click measure. Export the results or make a note of the DNA concentration and the 260/280 and 260/230 ratios.

SECTION: 2. DNA quantification
25. To quantify using the QuantiFluor ONE dsDNA kit, prepare DNA standards in Eppendorf tubes as shown in Table 1.
 
Add 200 µL of QuantiFluor dye to each well of a black microplate and add 1 µL of each standard dilution in duplicate or triplicate, 1 µL TE buffer as a negative control, or 1 µL of DNA. Gently pipette up and down or vortex to mix and incubate the plate at room temperature for 5 min. Place the plate in a spectrophotometer such as the BioTek Cytation 5 imaging reader and measure fluorescence at 504 nm excitation and 531 nm emmision. Calculate the DNA concentration according to the standard curve.
SECTION: 2. DNA quantification
26. To quantify using the Quant-iT dsDNA high-sensitivity kit, dilute the reagent 1:200 in buffer in a plastic centrifuge tube and invert to mix. Add 200 µL of this solution to each well of a black microplate. Add 10 µL of the provided standards or 10 µL of DNA to the wells. Gently pipette up and down or vortex to mix and incubate the plate at room temperature for 5 min. Place the plate in a spectrophotometer such as the BioTek Cytation 5 imaging reader and measure fluorescence at 502nm excitation and 523 nm emmision. Calculate the DNA concentration according to the standard curve.

SECTION: 3. Step 1 PCR
27. Order primers with the Illumina adaptor sequences attached at the 5' end according to Table 2 and 3.

 

 

SECTION: 4. PCR clean up
37. Transfer 20 µL of PCR product to a Millipore MultiScreen PCR plate and add 80 µL of TE buffer, pipette up and down to mix and avoid bubbles.
SECTION: 5. Step 2 PCR
41. Defrost and centrifuge primers at 1000 x g for 1 min. Prepare primer array plates for dual-indexing of samples by using a liquid handling robot such as Opentrons to transfer 20 µL of each forward and reverse indexing primer pair at 50 micromolar (µM) to 360 µL of molecular grade water in a 96-deep well block for a final primer concentration of 0.5 micromolar (µM). Transfer an aliquot of 5 µL from the master array plate to a separate plate for PCR, respecting plate positions. Seal all plates with an adhesive plate seal.
 

SECTION: 6. Normalisation
50. Use the Norgen NGS normalisation kit to normalise DNA to 5 ng/µL. Add 60 µL of buffer SK to20 µL of PCR product in the PCR plate, pipette up and down to mix and then transfer all to the 96-well normalisation plate mounted on top of a collection plate. Seal with an adhesive plate seal.
SECTION: 7. Gel extraction
60. Vacuum concentrate the library to approximately 30 ul.
SECTION: 4. PCR clean up
38. Place the plate onto a vacuum manifold and apply vacuum at   for 12 min, making sure the wells are completely empty before removing from the vacuum. Blot droplets.

SECTION: 4. PCR clean up
39. Add 35 µL of molecular grade water to each well and vortex at   for 10 min.

SECTION: 4. PCR clean up
40. Transfer cleaned PCR product to a PCR plate and store at 4 °C in a fridge short-term (up to 1 week) or in a freezer at -20 °C for long term storage.
SECTION: 5. Step 2 PCR
42. Defrost PCR reagents in Table 6 and gently vortex to mix before use. Defrost the cleaned step 1 PCR product and the aliquoted primer array plate and centrifuge at 1000 x g for 1 min. Clean the laminar flow hood with ethanol and then place all pipettes, pipette tips, reagent reservoirs, centrifuge tubes, plate seals and gloves in a laminar flow hood for UV-sterilisation.

SECTION: 5. Step 2 PCR
43. In the flow hood, combine the PCR reagents in the order presented in a centrifuge tube to form a master mix for the number of samples required according to Table 6. Add 5% to the number of samples the master mix is prepared for to account for pipetting error or loss. Gently invert to mix.

 

SECTION: 5. Step 2 PCR
44. Dispense the master mix into a reagent reservoir and transfer 18 µL to each well of the aliquoted primer array plate, taking care not to touch primers in the plate with the pipette tip.

SECTION: 5. Step 2 PCR
45. Add 2 µL of cleaned step 1 PCR product to each well and make a note of sample positions. Pipette up and down to mix. leave at least one well free of DNA as a negative PCR control, or add 2 µL of negative step 1 PCR blank. Seal with an adhesive plate seal.
SECTION: 5. Step 2 PCR
46. Centrifuge at 1000 x g for 1 min.
SECTION: 5. Step 2 PCR
47. Place the plate in a thermocycler and run the program in table 7.

 


SECTION: 5. Step 2 PCR
48.   for gel electrophoresis.
SECTION: 6. Normalisation
51. Centrifuge at 3200 x g for 2 min. Discard the flow through from the collection plate into a hazardous waste bottle.
SECTION: 6. Normalisation
52. Add 90 mL 100% ethanol to wash solution A, invert the bottle to mix. Remove the plate seal and add 400 µL of wash solution A with ethanol added to each well of the normalisation plate on the emptied collection plate. Replace plate seal.
SECTION: 6. Normalisation
53. Centrifuge at 3200 x g for 2 min. Discard the flow through from the collection plate into a hazardous waste bottle.
SECTION: 6. Normalisation
54.   to repeat the wash step. After the second wash step, centrifuge at 3200 x g for 15 min.

SECTION: 6. Normalisation
55. Place the normalisation plate on top of an elution plate, remove the plate seal and add 100 µL of elution buffer to each well.

SECTION: 6. Normalisation
56. Centrifuge at 200 x g for 1 min and then at 3200 x g for 5 min.
SECTION: 7. Gel extraction
61. Prepare a 2 % (v/v) agarose gel. For example, for a mini gel (up to 1 combs of 8 wells), combine 100 mLof 1X TBE buffer with 2 g agarose in a conical flask. Heat in a microwave until all of the agarose has dissolved, allow to cool to approximately 50 °C and add 5 mL GelRed or SybrSafe DNA stain. Mix well and pour into the gel tray with combs in place and secured in a gel caster. Allow to set for approximately 10 min. Once set, carefully remove the combs and transfer the gel within the gel tray to a gel tank and submerge in 1X TBE buffer.
SECTION: 7. Gel extraction
62. Add 10 µL of loading buffer to the library and transfer to a well in the gel. Add 5 µL of 100 Kb hyperladder to at least one well and run the gel at 90 V for 60 min .
SECTION: 7. Gel extraction
63. Place the gel on a UV light box and with a sterile scalpel, cut the library band from the gel and place in a pre-weighed Eppendorf tube. Weigh the Eppendorf after adding the gel slice to calculate the weight of the gel slice.
SECTION: 6. Normalisation
57. Pool normalised PCR product by plate into a centrifuge tube.
SECTION: 6. Normalisation
58. Quantify the plate pool with the Qubit dsDNA high sensitivity kit. Dilute stain 1:200 in buffer in a centrifuge tube. Add 190 µL of solution to a Qubit tube and add 10 µL sample or standard (scale for the number of samples required, including 2 standards and add 2 for pipetting error or loss). Vortex to mix. Incubate at room temperature for 2 min before measuring the DNA concentration of the standards and then of the samples on the Qubit fluorometer.
SECTION: 6. Normalisation
59. For all of the plate pools that will be sequenced on the same sequencing run, dilute an aliquot of each plate pool with elution buffer to achieve a normalised concentration in 50 µL. Pool all of the plate pools of each amplicon (taking into account sample numbers if each plate had a different number of samples) to form the normalised library (pool different amplicons separately).
SECTION: 7. Gel extraction
64. Use the Qiagen gel extraction MinElute kit to extract the purified library from the gel slice. Add 3 volumes of buffer QG to 1 volume of gel (100 mg gel = 100 µL) in the Eppendorf tube.

SECTION: 7. Gel extraction
65. Incubate at 50 °C for 10 min and invert the tube periodically. Make sure the gel has fully dissolved.

SECTION: 7. Gel extraction
66. Add 1 volume of isopropanol to the sample and invert to mix.
SECTION: 7. Gel extraction
67. Transfer up to 700 µL of the sample to a MinElute spin column in a collection tube and centrifuge at 17900 x g for 1 min. Discard the flow through into a hazardous waste bottle. Repeat until all of the sample has been filtered.
SECTION: 7. Gel extraction
68. Add 100% ethanol to buffer PE according to the label on the bottle. Add 750 µL buffer PE with ethanol added to the spin column in an emptied collection tube. Let the spin column stand for 5 min.

SECTION: 7. Gel extraction
69. Centrifuge at 17900 x g for 1 min. Discard the flow through into a hazardous waste bottle and repeat the centrifugation step.

SECTION: 7. Gel extraction
70. Place the spin column into an Eppendorf tube and add 100 µL of elution buffer. Let the spin column stand for 1 min.

SECTION: 7. Gel extraction
71. Centrifuge at 17900 x g for 1 min.

SECTION: 5. Step 2 PCR
49. Store at 4 °C in a fridge short-term (up to 1 week) or in a freezer at -20 °C for long term storage.
SECTION: 7. Gel extraction
72.   to quantify the gel extracted library. Aim for a concentration of 0.2-0.6 ng/ul and dilute with elution buffer if necessary.

SECTION: 8. Library preparation and sequencing
73. Calculate the molarity (nM) of each amplicon according to its length in bp and calculate the concentration of the library in nM using the Qubit reading. If multiple amplicons are to be sequenced on the same run, pool in an equimolar ratio (taking into account number of samples and amplicon molarity). Dilute to a final loading library concentration of 1000 picomolar (pM) if sequencing on the Illumina NextSeq platform with onboard denaturation, or to 400 picomolar (pM) if sequencing on the Illumina MiSeq platform with manual denaturation.
SECTION: 3. Step 1 PCR
28. Defrost PCR reagents in Table 2 and gently vortex to mix before use. Clean the laminar flow hood with ethanol and then place all pipettes, pipette tips, reagent reservoirs, centrifuge tubes, plates, plate seals and gloves in a laminar flow hood for UV-sterilisation.
SECTION: 3. Step 1 PCR
29. In the flow hood, combine the PCR reagents in the order presented in a centrifuge tube to form a master mix for the number of samples required according to Table 4. Add 5% to the number of samples the master mix is prepared for to account for pipetting error or loss. Gently invert to mix.
 

SECTION: 3. Step 1 PCR
30. Dispense the master mix into a reagent reservoir and transfer 48 µL to each well of a PCR plate.
SECTION: 3. Step 1 PCR
31. Add 2 µL of DNA to each well and make a note of sample positions. Pipette up and down to mix. leave at least one well free of DNA as a negative PCR control, or add 2 µL of negative extraction blank. Seal with an adhesive plate seal.
SECTION: 3. Step 1 PCR
32. Centrifuge at 1000 x g for 1 min.
SECTION: 3. Step 1 PCR
33. Place the plate in a thermocycler and run the relevant program for each primer in Table 5.

 

SECTION: 3. Step 1 PCR
34. Prepare a 1.5 % (v/v) agarose gel to visualise PCR product. For example, for a maxi gel (up to 4 combs of 52 wells), combine 250 mLof 1X TBE buffer with 3.75 g agarose in a conical flask. Heat in a microwave until all of the agarose has dissolved, allow to cool to approximately 50 °C and add 5 mL GelRed or SybrSafe DNA stain. Mix well and pour into the gel tray with combs in place and secured in a gel caster. Allow to set for approximately 20 min. Once set, carefully remove the combs and transfer the gel within the gel tray to a gel tank and submerge in 1X TBE buffer.
SECTION: 3. Step 1 PCR
35. Transfer 5 µL of PCR product to a microplate and add 1 µL of loading buffer to each well. Transfer each sample to the wells of the gel. Add 5 µL of 100 Kb hyperladder to at least one well per row and run the gel at 130 V for 45 min .
SECTION: 3. Step 1 PCR
36. Carefully place the gel on a gel imager such as the BioRad GelDoc imager to visualise the gel and determine if PCR amplification was successful and the PCR product is of the expected size according to the ladder.