TITLE: 16S rRNA gene Library Preparation Protocol
AUTHORS: dinesh.aggarwal, Katherine L Bellis, Josef Wagner, eh

[DESCRIPTION]
16S rRNA gene library preparation protocol including the following steps: MPure extraction of Bacterial DNA, Manual 16S indexed primer PCR, library clean up with beads, quibit DNA quantification, equimolar mix construction, agarose gel DNA concentration and gel clean up to produce a library ready for Illumina sequencing. 

[BEFORE_START]
Barcoded primers were reconstituted as instructed by the manufacturer to a stock concentration of 100mM.

[GUIDELINES]
Contamination of microbiome samples is a large scale problem, and all steps of this process should be optimised to avoid it. Undertake activity in a freshly cleaned MSC or PCR hood as far as possible. Keep samples closed, use new tips and seal plates well.

[STEPS]
SECTION: DNA Extraction using the MPBio MPure-12:
1. UV the MPure machine before use.
SECTION: DNA Extraction using the MPBio MPure-12:
3. Remove samples to be extracted from the freezer, to thaw in the fridge (4 °C). 
Once thawed vortex for 10 s. 
Proceed in MSC.
SECTION: DNA Extraction using the MPBio MPure-12:
5. Pipette mix and remove up to 800 µL from the storage tube and transfer to a labelled 1.5 ml Eppendorf tube

SECTION: DNA Extraction using the MPBio MPure-12:
6. Centrifuge tube for 10 min at 13200rpm.

SECTION: DNA Extraction using the MPBio MPure-12:
7. Remove and discard all bar 100 µLof the supernatant. Resuspend the bacterial pellet in the remaining liquid.

SECTION: DNA Extraction using the MPBio MPure-12:
9. Transfer the volume to a labelled lysis tube ( Lysing Matrix E, MP Biomedicals). Label the side of the tubes.
SECTION: DNA Extraction using the MPBio MPure-12:
10. Place lysis tubes in the ‘FastPrep Instrument’ and run for 40 s at speed 6000.

SECTION: DNA Extraction using the MPBio MPure-12:
11. Centrifuge tube for 10 minat 13500 rpm.

SECTION: DNA Extraction using the MPBio MPure-12:
13. Remove the supernatant (450 µL) from each lysis tube to a labelled blue sample tube from MPure bacterial DNA extraction kit. 

Place these in the metal rack, in the slots labelled “S” on the machine. 
SECTION: DNA Extraction using the MPBio MPure-12:
12. Set Up the MPure 12 Reagents and plasticware:

Place the reagent cartridge in the large flat block at the back, it should click into place, and the writing and release flap should be facing toward you.

The clear plastic mixing wells are clipped in front of these.

Then place the black plastic container set into the next slots, into the first of these place a plastic pin, in the third place a tip.

Put an open labelled elution tube in the closest holes in the metal rack (labelled E).
SECTION: DNA Extraction using the MPBio MPure-12:
14. Machine instructions: 
Select “START”, it will scan the setup from the sheet in the kit. 
Select the protocol : Mpure Bacterial DNA extraction kit with barcode: “OP02006”·       
Select sample volume 400ul with barcode ”SV0400”·       
Select elution volume 50ul with barcode “EV0050”·       
Press Enter to start the extraction 

The run time is around 1 hour. The machine will stall if an error develops, but will not stall if a tip is lost, or plasticware comes unclipped. 
SECTION: DNA Extraction using the MPBio MPure-12:
15. Remove elution tubes, checking a reasonable volume is present, and store these at -80 °Cin a labelled box.

SECTION: 16S rDNA PCR and Clean Up -Preparation:
16. Clean MSC or PCR hood with DNAZap.
SECTION: 16S rDNA PCR and Clean Up -Preparation:
17. Dilute primers to working concentration (10 millimolar (mM),) in NFW, if there are no pre-existing dilutions < 2 weeks old.

SECTION: 16S rDNA PCR and Clean Up -Preparation:
18. Aliquot diluted primers into labelled, linked PCR tubes. Freeze upright overnight if necessary.
SECTION: 16S rDNA PCR and Clean Up -Preparation:
19. Rack out the samples for the library into 12 x 8 rack according to use, complete plate plan, put on lid, label and store in -80°C. 
Remember to include:
- PCR negative control(s) from the relevant DNA extractions 
- positive control (mock community or bacterrial DNA)
- negative PCR control (nuclease free water). 
-  any diluted, or pre-spiked samples.
SECTION: 16S rDNA PCR :
20. Wipe down MSC with Distel/DNAZap, and then 80% ethanol. 
Wipe down all equipment with distel and put in the MSC. 
Close MSC, and UV.
SECTION: 16S rDNA PCR :
21. Defrost the reagents on ice.
SECTION: 16S rDNA PCR :
22. Add 14.25 µL nuclease free water to labelled master mix tubes.

SECTION: 16S rDNA PCR :
23. Add 5 µLQ5 buffer (Q5 High-Fidelity Polymerase Kit from New England Biolabs) to the master mix tubes. Mix before use, vortexing if salts have fallen from solution.

SECTION: 16S rDNA PCR :
24. Add 10 millimolar (mM) dNTP (0.5 µLper well) to the master mix tubes. Mix before use.

SECTION: 16S rDNA PCR :
25. Pipette mix the master mix, and keep on ice.
SECTION: 16S rDNA PCR :
26. Record reagent lot numbers on the library plate plan sheet. Try to use the same PCR reagents for all samples, if  possible.
SECTION: 16S rDNA PCR :
27. Add Q5 enzyme (5 µLper well) to individual master mix tubes immediately before use, keep on ice. Pipette mix.
SECTION: 16S rDNA PCR :
28. Prepare a 96 well plate for use, labelling and adding orientation points as necessary. Use the plate ice block from this point on.
SECTION: 16S rDNA PCR :
29. Pipette 20 μl of Master mix into each well of the 96 well plate. There is no need to change tips, but the buffer may bubble.
SECTION: 16S rDNA PCR :
30. 1.25 μl of each primer (501-508) can be transferred using a multichannel pipette in the vertical from the set of linked PCR tubes, swap tips if the plate or tubes are touched. Check each tip has taken up the correct volume.
SECTION: 16S rDNA PCR :
31. 1.25 μl of each primer (701-712) can be transferred using a multichannel pipette on the horizontal from a set of linked PCR tubes, tips should be changed after each use. Check each tip has taken up the correct volume.
SECTION: 16S rDNA PCR :
32. Apply a plate seal and move the plate, and it’s chiller unit into a second MSC if available.
SECTION: 16S rDNA PCR :
33. Retrieve the samples from the fridge, spray the closed rack into the MSC.
SECTION: 16S rDNA PCR :
34. Carefully remove the plate seal. Change gloves. 
SECTION: 16S rDNA PCR :
35. Add 2.5 μl of each sample DNA template into each well, according to your plate plan. Change tips every time and discard if they have touched anything before use.
SECTION: 16S rDNA PCR :
36. Return samples to the fridge.
SECTION: 16S rDNA PCR :
37. Apply a plate seal and use a roller and squeegee to ensure that the wells seal properly. If you find large amounts of loss from outer wells apply autoclave tape around the edges.
SECTION: 16S rDNA PCR :
38. Run this plate on a PCR machine with a heated lid.
 
Initially at 98 °C for 2minutes. 
32 cycles of 98 °C for 30 seconds, 50 °C for 30 seconds, 72 °C for 1 minute and 30
seconds.
Finally 72 °C for 5mins and hold at 4 °C.


SECTION: 16S rDNA PCR :
39. Wipe Down MSC and pipettes with DNAZap.
SECTION: 16S rDNA PCR :
40. Repeat steps 28-40 until the library has been prepared and run in triplicate.
SECTION: 16S rDNA PCR :
41. These plates can be frozen at -20 °C before clean up.

SECTION: Magnetic Bead Clean Up:
42.  Bring beads to Room temperature, prepare 80% ethanol and TRIS-HCl before beginning.

SECTION: Magnetic Bead Clean Up:
44. Combine the triplicate PCR products into 1 plate using a multichannel pipette.
SECTION: Magnetic Bead Clean Up:
45. Vortex the beads stock (Agencourt AMPure XP beads) for 30 seconds to ensure even dispersal. Pour into a trough.
SECTION: Magnetic Bead Clean Up:
46. Add an equal volume of beads per volume of PCR product (probably 75 µL) to each well, gently pipetting up and down x10, changing tips for every column.

SECTION: Magnetic Bead Clean Up:
47. Incubate at Room temperature for 5 min

SECTION: Magnetic Bead Clean Up:
48. Move the plate to the magnetic stand for 2 minuntil the supernatant has cleared.

SECTION: Magnetic Bead Clean Up:
49. Leaving the plate on the magnetic stand use a multichannel pipette to remove and discard the supernatant, changing tips every time.

If you disrupt the beads leave the well until the end to allow them to resettle. The beads should remain attached to the side of the well.
SECTION: Magnetic Bead Clean Up:
50. Leaving the plate on the magnetic stand wash the beads with 200 µLfresh 80% ethanol, using a multichannel pipette. 

SECTION: Magnetic Bead Clean Up:
52. Leaving the plate on the magnetic stand wash the beads with fresh 80% ethanol. Using a multichannel pipette add 200 µL of fresh 80% ethanol to each well.

SECTION: Magnetic Bead Clean Up:
54. Leave the PCR plate on the magnetic plate for 10 min to air dry.

SECTION: Magnetic Bead Clean Up:
55. Remove the plate from the magnetic stand. Using a multichannel pipette add 27.5 µLof TRIS-HCl pH8 into each well of the plate.

SECTION: Magnetic Bead Clean Up:
56. Pipette up and down x10 to re-suspend the beads, Some wells may need more flushing than others, ensure that the beads are fully resuspended.
SECTION: Magnetic Bead Clean Up:
57. 2 min Incubate the plate at room temperature for 2 min

SECTION: Magnetic Bead Clean Up:
58. Put the PCR plate on the magnetic stand for 2 min (or until the supernatant clears).

SECTION: Magnetic Bead Clean Up:
59. Using a multichannel pipette move 25 µL from each well into a new clean labelled PCR plate. Change tips between samples. Do not disturb the bead pellet .

SECTION: Magnetic Bead Clean Up:
60. Seal plate thoroughly. If freezing at -20 °C use aluminium foil. Keep at 4 °C overnight.

SECTION: Quantify PCR products and make Equimolar Mix:
61. Label Qubit tubes with the well names (Sample Tubes).


SECTION: Quantify PCR products and make Equimolar Mix:
63. Briefly vortex and spin down the samples.
SECTION: Quantify PCR products and make Equimolar Mix:
64. Wrap tape and foil around a 50ml Falcon Tube to ensure no light enters.
SECTION: Quantify PCR products and make Equimolar Mix:
65. Into the Falcon Tube, add Qubit HS Buffer (199 µL x (sample no.+2)) and Qubit HS Reagent (1 µL x (sample no.+2)), close and vortex well. This working solution is light reactive; ensure that the duration of light exposure is minimal.

SECTION: Quantify PCR products and make Equimolar Mix:
66. Add 190 µL of Working Solution to each of the Standard Tubes, vortex for 5 s and leave for 2 min. Ensure there are no bubbles and all liquid is at the bottom of the tube.

SECTION: Quantify PCR products and make Equimolar Mix:
67. Select dsDNA HS on the Qubit machine & calibrate using the 2 Standard Tubes. Note down the values for the Standards.
SECTION: Quantify PCR products and make Equimolar Mix:
68. Vortex the Working Solution Tube and add 198 µLnto a Sample Tube, then add 2 µL of the correct sample, vortex for 5 s and leave for 2 min. Ensure there are no bubbles in the Sample Tube and all liquid is at the bottom of the tube. 

SECTION: Quantify PCR products and make Equimolar Mix:
69. Place Sample Tube into the Qubit machine and record the DNA concentration.
SECTION: Quantify PCR products and make Equimolar Mix:
70. Repeat steps 68-69 for the remaining samples. Dilute any samples where the concentration is too high and repeat to get a value.
SECTION: Quantify PCR products and make Equimolar Mix:
71. Find the highest and lowest concentration of pooled PCR product & use this to decide the amount of DNA from each sample to be added to equimolar mix (ensure volume of lowest is possible (eg. 20 µL) and volume of largest is possible 1 µL). Remember to scale up any diluted samples.  

e.g.             Highest = 55.0μg/ml         Lowest = 6.2μg/ml 
55μg/ml  x 3 ul = 165μg              165 / 6.2 = 26.6μl
However, only 20μl any sample, so: 55 x 2 = 110μg              110 / 6.2 = 17.7μl)
SECTION: Quantify PCR products and make Equimolar Mix:
72. Once amount of DNA is calculated, use the following formula to work out volume of each sample to be added to equimolar mix: Volume (μl) = DNA amount (μg) / Sample Concentration (μg/ml). If the concentration of a sample is <2ng/μl, add all the sample volume to the equimolar mix to give the best chance of enough reads.
SECTION: Quantify PCR products and make Equimolar Mix:
73. Print list of needed volumes in 96 well format.
SECTION: Quantify PCR products and make Equimolar Mix:
74. Mix the correct volumes of each pooled PCR product to create the equimolar mix in a labelled, screw cap 2ml eppendorf. Add the largest volumes first. 
SECTION: Quantify PCR products and make Equimolar Mix:
75. Pipette mix and check that the total volume is correct.
SECTION: Quantify PCR products and make Equimolar Mix:
76. Split the equimolar mix volume in half. 
Store half of the equimolar mix at -20 °C in a o-ring screwcap tube.  

SECTION: Gel Purification: Removes Primer dimers and concentrates PCR products.
77. Mix the unfrozen half of the equimolar mix with loading dye as instructed by the dye information leaflet.
SECTION: Gel Purification: Removes Primer dimers and concentrates PCR products.
78. Make 1% agarose, 1x TBE gel with the nucleic acid gel stain of your choice, load into buffer tank and top up the buffer until it is level with the top of the gel. 

SECTION: Gel Purification: Removes Primer dimers and concentrates PCR products.
81. Add buffer to cover and run for approximately 90 min.

SECTION: Gel Purification: Removes Primer dimers and concentrates PCR products.
82. Use a UV transilluminator to visualize the bands. Cut out the bands with as little excess gel as possible. Wear suitable face and eye covering, and ensure any nearby workers are taking suitable precautions. Dispose of any sharps appropriately. 
SECTION: Gel Purification: Removes Primer dimers and concentrates PCR products.
83. Weigh as many 1.5ml tubes as you have bands, and note their weight. Place the bands in to the 1.5ml tubes. Weigh the tubes to estimate the weight of gel slice.
SECTION: Gel Purification: Removes Primer dimers and concentrates PCR products.
84. Add 10 µL of Membrane Binding Solution (Wizard SV Gel and PCR Clean Up Kit from Promega) per 10 mgof gel slice and vortex briefly.

Eg. tube + gel slice = 1.48g, tube = 1g, gel slice = 0.48g, add 480μl of Membrane Binding Solution.   
SECTION: Gel Purification: Removes Primer dimers and concentrates PCR products.
86. There will only be 1 collection tube used per Equimolar mix. Place a Minicolumn into a Collection Tube and pipette up to 700 µLof dissolved gel into the column.  Incubate for 1 minute. 

SECTION: Gel Purification: Removes Primer dimers and concentrates PCR products.
87. Centrifuge the SV Minicolumn at 13200 rpm for 1 min.

SECTION: Gel Purification: Removes Primer dimers and concentrates PCR products.
88. Discard the supernatant into a labelled liquid waste container and reinsert the SV Minicolumn into the Collection Tube.
SECTION: Gel Purification: Removes Primer dimers and concentrates PCR products.
89. Repeat steps 87-89 until no dissolved gel mixture remains ( if > than 700 µLof dissolved gel).

SECTION: Gel Purification: Removes Primer dimers and concentrates PCR products.
90. Add 700 µL of Membrane Wash Solution (add ethanol before use as instructed by kit).

SECTION: Gel Purification: Removes Primer dimers and concentrates PCR products.
91. Centrifuge at 13200 rpm for 1 min.

SECTION: Gel Purification: Removes Primer dimers and concentrates PCR products.
92. Discard the supernatant and reinsert the SV Minicolumn into the Collection Tube.
SECTION: Gel Purification: Removes Primer dimers and concentrates PCR products.
93. Add 500 µL of Membrane Wash Solution.

SECTION: Gel Purification: Removes Primer dimers and concentrates PCR products.
94. Centrifuge at 13200 rpm for 5 min
 
SECTION: Gel Purification: Removes Primer dimers and concentrates PCR products.
95. Discard the supernatant. Centrifuge the tube and minicolumn at 13200 rpm for 1 minute.

SECTION: Gel Purification: Removes Primer dimers and concentrates PCR products.
96. Transfer the SV Minicolumn to a 1.5ml tube and add 100 µLof Nuclease-Free Water directly onto the membrane, incubate for 1 minat RTP.

SECTION: Gel Purification: Removes Primer dimers and concentrates PCR products.
97. Centrifuge at 13200 rpm for 1 min.

SECTION: Gel Purification: Removes Primer dimers and concentrates PCR products.
98. Check the water has gone through the column and that the membrane is dry. If water remains on the matrix, repeat step 97.
SECTION: Gel Purification: Removes Primer dimers and concentrates PCR products.
99. Discard SV Minicolumn and split sample, for storage in a labelled screwcap tube with O-ring. Keep half as a spare and submit the other half for sequencing.
SECTION: DNA Extraction using the MPBio MPure-12:
8. Add 600 µLof BLB2 buffer (MPure Bacterial DNA Extraction Kit’, MP Biomedicals)and vortex to further resuspend.

SECTION: DNA Extraction using the MPBio MPure-12:
4. A negative control (e.g. nuclease free water), should be done for each kit (approx. every 50 samples) to show the presence of contaminants (particularly important in low biomass samples) and records should be kept to link this to the samples extracted, machine used, and kit information.
SECTION: Quantify PCR products and make Equimolar Mix:
62. Label two Qubit tubes 'S1' and 'S2' and place 10 µLof Qubit HS Standard 1 into the first and 10μl of Standard 2 into the second RTP.

SECTION: Gel Purification: Removes Primer dimers and concentrates PCR products.
80. Run for 10 minat 90V with the buffer level with the top of the gel. The equimolar mix should have moved into the gel.

SECTION: Gel Purification: Removes Primer dimers and concentrates PCR products.
79. Load the Equimolar mix into the gel, filling the well(s) as much as possible.
SECTION: Gel Purification: Removes Primer dimers and concentrates PCR products.
85. Place tube into a heat block at 55 °C until gel dissolves completely. You may want to vortex during this incubation to hasten the dissolving.

SECTION: Magnetic Bead Clean Up:
43. Defrost plates on ice and spin down before opening. Change gloves.
SECTION: Magnetic Bead Clean Up:
51. Incubate on the magnetic plate for 30 s. Carefully remove and discard the supernatant into a waste container.

SECTION: Magnetic Bead Clean Up:
53. Incubate on the magnetic plate for 30 s.Carefully remove and discard the supernatant into a waste container. Use a P20 with fine tips to remove any residual ethanol.

SECTION: DNA Extraction using the MPBio MPure-12:
2. Clean MSC with DNAZap.