TITLE: Library preparation protocol to sequence V3-V4 region of 16S rRNA to run in Illumina MiSeq platform
AUTHORS: Somasundhari Shanmuganandam, Benjamin Schwessinger, Robyn Hall

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is an optimised protocol for 16S library preparation of V3-V4 region for sequencing through Illumina MiSeq platform (2 x 300 bp V3 chemistry). This protocol uses Platinum™ SuperFi™ PCR Master Mix instead of 2x KAPA HiFi HotStart ReadyMix given in Illumina 16S protocol. This polymerase master mix has lower error rate than KAPA, making it more suitable for sequencing.  </div></div>

[STEPS]
?. [Stage 1 PCR amplification]
The PCR reaction setup is given below  AB1ReagentsVolume (µl) for 25µl reaction22X Platinum™ SuperFi™ PCR Master Mix12.5 µL3Water, nuclease-free Upto 25 µl410 µM forward primer 1.25 µL510 µM reverse primer 1.25 µL6Template DNA4.6 ng Add reagents and DNA in a sterilised PCR tube in the order given above
AB1ReagentsVolume (µl) for 25µl reaction22X Platinum™ SuperFi™ PCR Master Mix12.5 µL3Water, nuclease-free Upto 25 µl410 µM forward primer 1.25 µL510 µM reverse primer 1.25 µL6Template DNA4.6 ng
?. [Stage 1 PCR amplification]
PCR cycle conditions:Initial denaturation at 98°C for 30 sec 25 cycles of :98°C for 10 sec55°C for 15 sec72°C for 30 secFinal extension at 72°C for 10 min
?. [Stage 1 Clean up]
Bring the AMPure XP beads to room temperature.
?. [Stage 1 Clean up]
Prepare two Axygen plate with each well containing 200 μl of 80% ethanol
?. [Stage 1 Clean up]
Using a multichannel pipette set to 20 μl, transfer the entire Amplicon PCR product to the Axygen plate.  Change tips between samples.
?. [Stage 1 Clean up]
Vortex the AMPure XP beads for 30 seconds to make sure that the beads are evenly dispersed.
?. [Stage 1 Clean up]
Add 20 μl of AMPure XP beads (1:1 ratio) to each well on the plate
?. [Stage 1 Clean up]
Gently pipette entire volume up and down 10 times using pipette
?. [Stage 1 Clean up]
Incubate at room temperature without shaking for 5 minutes.
?. [Stage 1 Clean up]
Place a sheath on the coppin device (a device shaped like a 96 well plate with magnets attached at each well similar to 96-well Microtiter Plate Magnetic Separation Rack) and insert into the plate wells containing PCR product
?. [Stage 1 Clean up]
Allow beads to bind to the coppin device for 2 mins and use this device to transfer the beads to first ethanol plate.
?. [Stage 1 Clean up]
Hold in ethanol for 30 seconds
?. [Stage 1 Clean up]
Transfer beads to second ethanol half plate
?. [Stage 1 Clean up]
Hold in ethanol for 30 seconds
?. [Stage 1 Clean up]
Remove beads from ethanol using coppin device and invert it on the bench so that the beads are facing up.
?. [Stage 1 Clean up]
Air dry at room temperature for 10 minutes (still on coppin device)
?. [Stage 1 Clean up]
Using a multichannel pipette, add 52.5 μl of 10 mM Tris pH 8.5 (Buffer EB) to each well of the Amplicon PCR plate. Cover when not in use.
?. [Stage 1 Clean up]
With air dried bead still on the device, insert sheath into the well containing Tris and remove the coppin device whilst leaving the sheath in the solution
?. [Stage 1 Clean up]
Resuspend beads by gently swishing the sheath in the Tris. If beads don’t come off, use magnetic base to remove the stuck beads and remove the plate immediately.
?. [Stage 1 Clean up]
Incubate at room temperature for 2 minutes without shaking
?. [Stage 1 Clean up]
Put fresh sheath on Coppin device and insert into Tris for 2 mins
?. [Stage 1 Clean up]
Remove and discard the beads and the sheath.
?. [Stage 1 Clean up]
Using a multichannel pipette, remove the cleaned up product from the well and place it into PCR tube
?. [Stage 1 Clean up]
Run 5 µL of cleaned-up product in 1% agarose gel for 20 mins at 100V to check the size of the band (~500 bp)
?. Run 5 µL of PCR product in 1% agarose gel for 20 mins at 100V to check the size of the band (~460 bp)
?. [Stage 2 PCR amplification]
Nextera XT Index Kit (N7XX and S5XX) is used for the following set-up to add barcodes PCR reaction setup is given below  AB1Reagents Volume (50 µl)2Platinum™ SuperFi™ PCR Master Mix25 µl3Water, nuclease-freeupto 50 µl42µM of index (each)5 µl5Cleaned up DNA5 µl
AB1Reagents Volume (50 µl)2Platinum™ SuperFi™ PCR Master Mix25 µl3Water, nuclease-freeupto 50 µl42µM of index (each)5 µl5Cleaned up DNA5 µl
?. Cycle conditions for the above reavtion volume is Initial denaturation at 98°C for 30 sec 8 cycles of :98°C for 10 sec55°C for 15 sec72°C for 30 secFinal extension of 10 min at 72°C.
?. [Stage 2 PCR clean up]
Follow step 4 to step 25 but use 45 μl of AMPure XP in step 8 and 27.5 μl of 10 mM Tris pH 8.5 in step 18.
?. Run 5 µL of PCR product in 1% agarose gel for 20 mins at 100V to check the size of the band (~550 bp)
?. [Stage 2 PCR clean up]
Run 5 µL of PCR product in 1% agarose gel for 20 mins at 100V to check the size of the band. Run it next to stage 1 clean up product to check whether the product size is increased by addition of barcodes
?. [Quantification of stage 2 cleaned up product]
Quantify the stage 2 PCR product in Qubit high sensitivity assay
?. [Quantification of stage 2 cleaned up product]
Run tapestation on selection of samples to check the length
?. [Quantification of stage 2 cleaned up product]
Pool equimolar volume of each sample together to get a final concentration of 4 nM (1.17 ng /μl)
?. [Quantification of stage 2 cleaned up product]
Qubit the pooled sample using high sensitivity assay to ensure the concentration is 4 nM (1.17 ng /μl)
?. [Preparation of library for loading ]
Thaw MiSeq reagent cartridge in 25°C water bath
?. [Preparation of library for loading ]
Thaw the HT1 reagent and pooled library at room temperature and store it in ice box
?. [Preparation of library for loading ]
Prepare a fresh dilution of 0.2N NaOH (800 μl of dH20 + 200  μl of 1N NaOH)
?. [Preparation of library for loading ]
Combine 5 μl of pooled library with 5 μl of 0.2N NaOH Note: Work quickly from this step forward
?. [Preparation of library for loading ]
Vortex briefly and centrifuge for 1 min at 300 g.
?. [Preparation of library for loading ]
Incubate for 5 mins at room temperature (start as soon as centrifuge stops)
?. [Preparation of library for loading ]
Immediately add 990 μl of pre-chilled HT1 to denatured DNA. Place in ice until needed. This gives 20 pM of denatured library
?. Prepare 20 pM dilution of PhiX
?. Dilute denatured DNA to a final concentration of 9.5 pM by taking 285 μl of denatured library and adding 315 μl of pre-chilled HT1 (final volume 600 μl )
?. Invert several times to mix and pulse centrifuge. Place it in ice
?. Dilute 20 pM denatured PhiX to same concentration as final library. Can be performed by adding 285 μl of 20 pM denatured PhiX with 315 μl pre-chilled HT1
?. Invert several times to mix and pulse centrifuge. Place it in ice
?. Combine 60 μl of denatured, diluted PhiX with 540 μl of denatured diluted library
?. Incubate the combined library at 96 °C for 2 min
?. Invert several times to mix and place on ice immediately
?. Invert reagent cartridge and buffer bottle to mix
?. Load sample into reagent cartridge
?. Wash flow cell with water, dry and wash with ethanol
?. Load flow cell, reagent cartridge, buffer bottle, waste bottle into MiSeq. Ensure sample sheet is loaded. Start the run