TITLE: Illumina Amplicon Sequencing using two step PCR
AUTHORS: Lindsay Kate Newbold, Joetay, Jonathan Warren, Daniel S Read, kerry.walsh, Amy Thorpe

[DESCRIPTION]
This document is designed to provide the researcher with all of the information required to undertake two step amplicon sequencing using the Illumina MiSeq platform at UKCEH-Wallingford.

The aim is to provide a framework upon which the researcher may choose to add their own modifications to suit novel applications.

[STEPS]
SECTION: Nextera index plates- Prepared prior to Step 1 PCR
1. Order indexing primers direct from oligo manufacturer (For example IDT, Sigma genosys or MWG) suspended in water 0.5 µM scale, Desalt purification and10 µM  concentration. These indexing primers consist of: an Illumina Nextra adapter  i5 (Forward primer) AATGATACGGCGACCACCGAGATCTACAC or i7 (reverse primer) CAAGCAGAAGACGGCATACGAGAT, Unique 8bp barcode sequence and pre-adapter i5 TCGTCGGCAGCGTC (F) or i7 GTCTCGTGGGCTCGG (R). Barcode design when demultiplexed allows for the unique assignment of an individual sequence to sample.  Figure 1 gives a graphical representation of how 16F, and 24R barcodes sequences are allocated to give 384, unique barcode combinations. Full barcode sequence is given in attached document.
   
  



SECTION: Nextera index plates- Prepared prior to Step 1 PCR
2. Prepare index plate ‘masters’ mannually allocating 250 µL of each diluted primer stock 10 micromolar (µM) per well  as per plate design detailed above into Deep-Well microtiter plates. Alternatively this can be done using a liquid handling Robot. 

SECTION: Nextera index plates- Prepared prior to Step 1 PCR
3. Make PCR plate clones of the ‘master’ plates, each well containing 5ul of mixed primer, label, seal and store at -20 for future use.
SECTION: Step I- Amplicon PCR with modified primers
5. Step 1 Amplicon primers consist of a pre adapter, sequencing primer and specific locus primer. Target primers are designed as per Table 1:

 


SECTION: Step I- Amplicon PCR with modified primers
6.  Using column ‘Combined sequence’ given in Table 1, order primers direct from oligo manufacturer (For example IDT, Sigma genosys or MWG) suspended in water 0.5 µM scale, Desalt purification and100 µM  concentration. 
SECTION: Step I- Amplicon PCR with modified primers
47. Use PCRcalcs.xlsx to calculate the amount of reagents require – add 10% to the number of your samples
SECTION: Step I- Amplicon PCR with modified primers
7.  
ABCReagentPer SamplePer PlateMolecular Grade Water40.5 ul4050 ul5 X Buffer1 ul100 ul10 mM dNTP's1 ul100 ulQ5 Taq Polymerase0.5 ul50 ulPrimers from primer barcode  array plate5 ul500 ulDNA Template (at  ~10 ng/ml)2 ul200 ul
 
SECTION: Step I- Amplicon PCR with modified primers
8. All reagents from above Table 2 (except template DNA) are added to a 5ml Tube (or equivalent) to form a  PCR Mastermix.
Mastermix is prepared on ice, using filter tips and under sterile conditions in the PCR hood. 

Once Mastermix has been made add 48 µL  of Mastermix to each well of a 96 well PCR plate using a multichannel pipette (accurate for volumes between 100 µL -  300 µL ).

We recommend the use of :

Axygen™ 96-Well Low Profile PCR Microplates (SKU AXY-PCR-96-LP-FLT-C) 
https://www.thistlescientific.co.uk/product/96-well-polypropylene-flat-top-pcr-microplate-low-profile-no-skirt-clear-nonsterile/

and 
 

Add  2 µL  of standard dilution from step 4 to respective position in the mastermix plate. This ensure's full traceablity when samples are demultiplexed during the bioinformatics steps performed post sequencing. It is therefore imperitive that experimentors make a note of which sample is added to each well of the PCR Mastermix plate.


SECTION: Step I- Amplicon PCR with modified primers
9. Seal plates with PCR film, and centrifuge, to concentrate sample.

We recommend the use of: 
 
https://www.thermofisher.com/order/catalog/product/AB0558


SECTION: Step I- Amplicon PCR with modified primers
10. Initiate PCR reaction on Thermal cycler
We recommend the BIORAD C1000 touch thermal cyle https://www.bio-rad.com/en-uk/product/c1000-touch-thermal-cycler?ID=LGTW9415 

PCR programme for 16S, 18S and ITS:
Denature 95 °C for 02:00m 
Denature 95 °C 00:15s 
Anneal 55 °C (16S) 52 (ITS) for 00:30s 
Extension72 °C for 00:30s 
Repeat ii. – iv. 30 cycles 
Final extension 72 °C for 10:00m 

PCR programme for rbcl:
Denature 95 °C for 02:00m 
Denature 95 °C 00:20s 
Anneal 55 °C  for 00:45s 
Extension 72 °C for 00:60s 
Repeat ii. – iv. 35 cycles 
Final extension 72 °C for 5:00m 

SECTION: PCR Clean-up
12. 
Clean-up Amplicon PCR step I using ZymoZR-96 kit (standard Manufacturers protocol), again using filter tips and utmost care throughout as samples are not indexed.
https://zymoresearch.eu/collections/dcc-pcr-purification-kits/products/zr-96-dna-clean-up-kit

Alternatively use Multiscreen-PCR96 Filter plate under standard manufacturers protocol if liquid handling robot and vacuum manifold are available.

https://www.merckmillipore.com/GB/en/product/MultiScreen-PCR96-Filter-Plate,MM_NF-MSNU03050
SECTION: Step II - Nextera indexing PCR
13. Use  1-10 µL  Cleaned PCR product as template for Step II- Indexing PCR (Table 3). Water added to PCR master mix is adjusted to make each reaction total  25 µL .

 
ABCReagentPer SamplePer PlateMolecular Grade Water40.5 ul4050 ul5 X Buffer1 ul100 ulHigh GC Buffer1 ul100 ul10 mM dNTP's1 ul100 ulQ5 Taq Polymerase0.5 ul50 ulPrimer F0.5 ul50 ulPrimer R0.5 ul50 ulDNA Template (at  ~10 ng/ml)2 ulAdded separately
 
SECTION: Step I- Amplicon PCR with modified primers
4. Make a standardised dilution plate of template DNA from DNA extraction process (See Extraction protocol). DNA concentration is determined through either Nanodrop or Qubit BR assay of raw extract, and then each extract is diluted to a standardised concentration (this is subject to extraction success, but shouldn't be below 5ng/ul to allow for effcient PCR), using the equation:

Desired Concentration (ng/ul)    
________________________________________        X Final desired volume = Amount of DNA to add
Raw extract Concentration (ng/ul)

To work out the amount of water its  :  Final desired volume - Amount of DNA to add

SECTION: Gel Electrophoresis
11. Verify PCR products using gel electrophoresis. 
SECTION: Gel Electrophoresis
11.1. Prepare the appropriate electrophoresis tank and casting tray. The number and type of gels required will depend on the number of samples.
  
  
 
SECTION: Gel Electrophoresis
11.2. To make a 1 % (v/v)agarose gel :  For a Midi Gel,  measure 1g of molecular grade agarose powder into a glass pyrex beaker and make up to 100 mL using 1 X TBE buffer. Heat in microwave to dissolve for approx 2 min. When no agarose crystals are visable, cool the gel in a sink filled with a little water, to just above room temperature,. Cooling in this way will take atleast 2 min. As a rule of thumb if you're able to hold the base of the beaker without discomfort the gel is ready.

SECTION: Gel Electrophoresis
11.3. To a 100ml mix add 1 μl ethidium bromide, or 5 μl Gel Red 
Swirl to mix and pour into gel casting tray set up as below. 


  
SECTION: Gel Electrophoresis
11.4. Place combs directly into the casting tray (in above tray line up comb ends with 2nd notch and push down). If bubbles are present these can be pushed out of gel using combs before final positioning and tightening lever.

Leave gel to set for at least 45 min.

SECTION: Gel Electrophoresis
11.5. Whilst gel is setting, prepare samples using 5 µL  PCR amplicon to 1 µL Bromophenol blue gel loading buffer.
https://www.thermofisher.com/order/catalog/product/10816015#/10816015

SECTION: Gel Electrophoresis
11.6. When set load loosen casting lever and place tray and gel into Gel tank filled with 1X TBE buffer to fill line. Remove combs (doing this in the buffer prevents bubbles forming in wells). Add 5 µL Hyper ladder 1KB https://www.bioline.com/hyperladder-1kb.html (or similar) to first well of agarose gel by placing tip into well and gently depressing pipette control, whilst slowly removing tip from well. Repeat proceedure with samples, each being placed in its an individual well.
SECTION: Gel Electrophoresis
11.7. When all samples are loaded put on gel tank lid, and plug in cables (red to red, black to black). Set gel to run for 45 min at 90 Volts. 
SECTION: Gel Electrophoresis
11.8. When run complete image gel using gel image system (eg  https://www.bio-rad.com/en-uk/product/gel-doc-xr-gel-documentation-system?ID=O494WJE8Z).

A successful PCR should look similar to below.

  

SECTION: Normalisation
15. 
Resultant PCR products are normalised using the Norgen NGS Normalization 96-Well Kit – 61900 under standard manufacturers protocol:
UK distributers
 https://geneflow.co.uk/product/ngs-normalization-96-well-kit-61900/
SECTION: Step II - Nextera indexing PCR
14. PCR programme :
Denature 95 °C for 02:00m 
Denature 95 °C for 00:15s 
Anneal 55 °C for 00:30s 
Extension 72 °C for 00:30s 
Repeat to total 8 cycles 
SECTION: Normalisation
16. Use ~  5 µL  normalised PCR product from each sample to form a pooled library. 
SECTION: Normalisation
17. Vacuum concentrate to a volume of approx   30 µL  pooled normalised plate library.
We reccomend:
https://www.eppendorf.com/gb-en/eShop-Products/Centrifugation/Concentrator/Concentrator-plus-p-PF-25748
SECTION: Normalisation
17.1. Whilst Vacuum concentration is occuring prepare a 50ml 1% Agarose gel, with a wide comb.
SECTION: Normalisation
18. Add 5 µL loading buffer to concentrate and run for 45 min at 90 Volts. 
SECTION: Normalisation
19. Extract DNA from agarose Gel using QIAquick Gel Extraction Kit - QIAGEN under manufactuers standard protocol. Samples eluted in  30 µL buffer EB.
SECTION: Normalisation
18.1. Excise resultant band using sterile scapel, and visualised on blue light Led trans illuminator

https://www.smobio.com/shop/product/ve0100-b-boxtm-blue-light-led-epi-illuminator-419
SECTION: Quantification
20. Gel Purified libraries are quantified using Invitrogen™ Qubit™ 1X dsDNA High Sensitivity (HS) quantification kit 
https://www.fishersci.co.uk/shop/products/qubit-1x-dsdna-hs-br-assay-kits/15850210
Under manufacturers protocol and companion qubit fluorometer 
https://www.fishersci.co.uk/shop/products/qubit-4-fluorometer-wifi/16223001#?keyword=Qubit%204%20flurometer

SECTION: Quantification
21. Calculate Molar concentration using the https://www.bioline.com/media/calculator/01_07.html
ITS Amplicon Size         488   bp
18S Amplicon Size        533   bp
16S Amplicon Size        450   bp
rbcl Amplicon Size        533   bp

If using multiple libraries mix equal concentrations of each ITS arrays:  eg. ITSA+B.

Your libray is now ready for sequencing!
SECTION: Sequencing on V2 or V3 Miseq
22. Please note that much of the following is an ammended version of the SChloss lab SOP, therefore their work should always be referenced.

Remove a V3  600 cycle reagent cartridge from the-20 °C freezer. Place in room temperature water bath for one hour. Place Hyb buffer tube in4 °Cfridge. While reagent cartridge is thawing, perform steps 16-20

SECTION: Sequencing on V2 or V3 Miseq
23. Prepare fresh 0.1 Molarity (M) NaOH.

SECTION: Sequencing on V2 or V3 Miseq
24. To a 1.5 mL tube, add 10 µL of library and 10 µLof 0.1 Molarity (M)NaOH. To a separate tube add 2 µL PhiX, 3 µL PCR grade water, and 5 µL of 0.1 Molarity (M) NaOH. Pipette to mix. Note: NaOH concentration on the flow cell must remain under 0.001M. 

SECTION: Sequencing on V2 or V3 Miseq
25. Allow the tubes to incubate at room temperature for 5 min. Immediately add 980 µL of ice-cold Hyb to the library tube, and 990 µLHyb to the PhiX tube.

 Note: the resulting 20pM PhiX can be frozen and used for subequent runs.

SECTION: Sequencing on V2 or V3 Miseq
26. Use Hyb to further dilute both the library and PhiX to 6pM for a v3 kit. 
See example below:
            a. (1.45 nM library x 10 ul) + (0.1N NaOH x 10 ul) + 980 ul Hyb = 14.5pM Lib, 0.002N NaOH
            b. (14.5pM lib x 275.86 ul) + 724.14 ul Hyb = 4.0pM lib, 0.00055N NaOH 
            c. [(10nM PhiX x 2 ul) + 3 ul H2O] + (0.2N NaOH x 5 ul) + 990 ul Hyb = 20pM PhiX, 0.001N NaOH
            d. (20pM PhiX x 200 ul) + 800 ul Hyb = 4.0pM PhiX, 0.0002N NaOH
            e. (4.0pM Lib x 900 ul) + (4.0pM PhiX x 100 ul) = solution loaded
            f. Solution loaded is 4.0pM overall with a 3.6pM Library concentration, 0.4pM PhiX concentration, and 0.000515N NaOH
SECTION: Sequencing on V2 or V3 Miseq
27. For a 10% PhiX run, combine900 µL of 6 picomolar (pM) Library and 100 µL PhiX in a final tube. 
Vortex. At this final stage heat to 96 °Cfor 2 min. Then cool immediately on ice.

SECTION: Sequencing on V2 or V3 Miseq
28. When the reagent cartridge has thawed, dry bottom with paper towel. Invert the cartridge repeatedly to check each well is thawed. This also serves to mix the reagents. Place in Fridge
SECTION: Sequencing on V2 or V3 Miseq
29. Load 600 ul of the final Libary/PhiX solution into well 17 on the reagent cartridge.
SECTION: Sequencing on V2 or V3 Miseq
30. Unbox flow cell and PR2 bottle.Thoroughly rinse the flow cell with Milli-Q water. Carefully dry by blotting with lint free wipes (Kimwipes). Give special attention to the edges and points of intersection between the glass and plastic. Wet a new wipe with 100% alcohol and wipe the glass on both sides avoiding the rubber intake ports.Visually inspect the flow cell to ensure there are no blemishes, particles, or fibers on the glass. Follow on screen instructions to load the flow cell, reagent cartridge, and PR2 bottle. Empty and replace the waste bottle.
SECTION: Sequencing on V2 or V3 Miseq
31. Transfer reagent cartridge, flow cell, PR2 bottle, and flash drive with the sample sheet to the MiSeq. Open Sample Sheet from the flash drive in the MiSeq Local run manager software and queue run. Ensure the machine recognizes the correct sample sheet and the run parameters are correct.
SECTION: Sequencing on V2 or V3 Miseq
32. Wait for the MiSeq to perform its pre-run checks, and press start. NOTE: If the pre-run check fails, try wiping down the flow cell again.
SECTION: Sequencing on V2 or V3 Miseq
32.1. Ideal parameters for a 90% run:
  a. Cluster density 1000-1100k/mm2 for v3 kits 
  b. >85% clusters passing filter 
  c. 10% aligned (amount of PhiX) 
  d. No spikes in corrected intensity plot
  e. All indices identified following index reads 
  f. Final >Q30 score of >70%
SECTION: Sequencing on V2 or V3 Miseq
32.2. When run complete, perform post run wash as per manufacturers recommendations.