Oct 20, 2023
Version 1
Illumina Amplicon Sequencing using two step PCR V.1
Forked from a private protocol
This protocol is a draft, published without a DOI.
- Lindsay Kate Newbold1,
- Joe Taylor1,
- Jonathan Warren1,
- Daniel S Read1,
- Kerry Walsh1,
- Amy Thorpe1
- 1UK Centre for Ecology & Hydrology
Protocol Citation: Lindsay Kate Newbold, Joe Taylor, Jonathan Warren, Daniel S Read, Kerry Walsh, Amy Thorpe 2023. Illumina Amplicon Sequencing using two step PCR. protocols.io https://protocols.io/view/illumina-amplicon-sequencing-using-two-step-pcr-cn4vvgw6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 08, 2023
Last Modified: October 20, 2023
Protocol Integer ID: 76661
Funders Acknowledgement:
Environment agency
Abstract
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.
Nextera index plates- Prepared prior to Step 1 PCR
Nextera index plates- Prepared prior to Step 1 PCR
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.
Figure 1: An illustration of Dual-index primer pair allocation
Array Plate description.xlsx 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.
Make PCR plate clones of the ‘master’ plates, each well containing 5ul of mixed primer, label, seal and store at -20 for future use.
Step I- Amplicon PCR with modified primers
Step I- Amplicon PCR with modified primers
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
Step 1 Amplicon primers consist of a pre adapter, sequencing primer and specific locus primer. Target primers are designed as per Table 1:
| A | B | C | D | E | F | G | |
| Source reference | Universal primer name | Target Group | pre-adapter | Sequencing primer sequence | Specific locus primer | Combined sequence | |
| Walters et al (2015) https://journals.asm.org/doi/10.1128/mSystems.00009-15 | 515F | Bacteria | TCGTCGGCAGCGTC | AGATGTGTATAAGAGACAG | GTGYCAGCMGCCGCGGTAA | TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTGYCAGCMGCCGCGGTAA | |
| Walters et al (2015) https://journals.asm.org/doi/10.1128/mSystems.00009-15 | 806R | Bacteria | GTCTCGTGGGCTCGG | AGATGTGTATAAGAGACAG | GGACTACNVGGGTWTCTAAT | GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGACTACNVGGGTWTCTAAT | |
| Ihrmark et al (2012) https://doi.org/10.1111/j.1574-6941.2012.01437.x | ITS7F | Fungi | TCGTCGGCAGCGTC | AGATGTGTATAAGAGACAG | GTGARTCATCGAATCTTTG | TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTGARTCATCGAATCTTTG | |
| Ihrmark (2012) https://doi.org/10.1111/j.1574-6941.2012.01437.x | ITS4R | Fungi | GTCTCGTGGGCTCGG | AGATGTGTATAAGAGACAG | TCCTCCGCTTATTGATATGC | GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTCCTCCGCTTATTGATATGC | |
| Mangot et al (2012) https://doi.org/10.1111/1462-2920.12065 | NSF563 | Eukaryotes | TCGTCGGCAGCGTC | AGATGTGTATAAGAGACAG | CGCGGTAATTCCAGCTCCA | TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCGCGGTAATTCCAGCTCCA | |
| Mangot et al (2012) https://doi.org/10.1111/1462-2920.12065 | NSR951 | Eukaryotes | GTCTCGTGGGCTCGG | AGATGTGTATAAGAGACAG | TTGGYRAATGCTTTCGC | GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTTGGYRAATGCTTTCGC | |
| Kelley et al (2018) ISBN: 978-1-84911-406-6 | rbcL-646F | Diatoms | TCGTCGGCAGCGTC | AGATGTGTATAAGAGACAG | ATGCGTTGGAGAGARCGTTTC | TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGATGCGTTGGAGAGARCGTTTC | |
| Kelley et al (2018) ISBN: 978-1-84911-406-6 | rbcL-998R | Diatoms | GTCTCGTGGGCTCGG | AGATGTGTATAAGAGACAG | GATCACCTTCTAATTTACCWACAACTG | GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGATCACCTTCTAATTTACCWACAACTG |
Table 1: Step I amplicon primers employed during for the amplication of biofilm communities
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.
| A | B | C | |
| Reagent | Per Sample | Per Plate | |
| Molecular Grade Water | 40.5 ul | 4050 ul | |
| 5 X Buffer | 1 ul | 100 ul | |
| High GC Buffer | 1 ul | 100 ul | |
| 10 mM dNTP's | 1 ul | 100 ul | |
| Q5 Taq Polymerase | 0.5 ul | 50 ul | |
| Primer F | 0.5 ul | 50 ul | |
| Primer R | 0.5 ul | 50 ul | |
| DNA Template (at ~10 ng/ml) | 2 ul | Added separately |
Table 2: 1 Step PCR Reagents in the order in which they should be added. Note that Template is added separately after PCR Mastermix plate has been prepared.
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)
and
Equipment
Research Plus Multichannel pipette
NAME
Multichannel Pipette
TYPE
Eppendorf
BRAND
3125000052
SKU
LINK
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.
Seal plates with PCR film, and centrifuge, to concentrate sample.
We recommend the use of:
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
Gel Electrophoresis
Gel Electrophoresis
Verify PCR products using gel electrophoresis.
Prepare the appropriate electrophoresis tank and casting tray. The number and type of gels required will depend on the number of samples.
A Mini-sub Cell GT Horizontal Electrophoresis Tank. Can run up to 28 samples + two ladder lanes. The 7 x 10 cm casting tray requires 50 ml agarose mix.
A Wide Mini-sub Cell GT Horizontal Electrophoresis Tank (Midi Tank). Can run up to 58 samples + two ladder lanes. The 15 x 10cm casting tray requires 100ml agarose mix.
Sub-Cell 96 GT Horizontal Electrophoresis Tank. Can run up to 100 samples + two ladder lanes. The 25 x 15 cm casting tray requires 250ml agarose mix.
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 00:02:00 . 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 00:02:00 . As a rule of thumb if you're able to hold the base of the beaker without discomfort the gel is ready.
4m
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.
Biorad gel caster. To use push two casting walls together and tighten using lever.
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 00:45:00 .
45m
Whilst gel is setting, prepare samples using 5 µL PCR amplicon to 1 µL Bromophenol blue gel loading buffer.
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.
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 00:45:00 at 90 Volts.
45m
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.
Figure 4: Result of ITS2 PCR.
PCR Clean-up
PCR Clean-up
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.
Alternatively use Multiscreen-PCR96 Filter plate under standard manufacturers protocol if liquid handling robot and vacuum manifold are available.
Step II - Nextera indexing PCR
Step II - Nextera indexing PCR
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 .
| A | B | C | |
| A | B | C | |
| Reagent | Per Sample | Per Plate | |
| Molecular Grade Water | 7.25 ul | 725 ul | |
| 5 X Buffer | 5 ul | 500 ul | |
| High GC Buffer | 5 ul | 500 ul | |
| 10 mM dNTP's | 0.5 ul | 50 ul | |
| Q5 Taq Polymerase | 0.25 ul | 25 ul | |
| Primer Array mix | 5 ul | Added separately | |
| PCR Template | 2 ul | Added separately |
Table 3: Step II PCR Reagents in the order in which they should be added. Note that Template and primer mix are added separately after PCR Mastermix plate has been prepared.
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
Normalisation
Normalisation
45m
45m
Resultant PCR products are normalised using the Norgen NGS Normalization 96-Well Kit – 61900 under standard manufacturers protocol:
UK distributers
Use ~ 5 µL normalised PCR product from each sample to form a pooled library.
Vacuum concentrate to a volume of approx 30 µL pooled normalised plate library.
We reccomend:
Whilst Vacuum concentration is occuring prepare a 50ml 1% Agarose gel, with a wide comb.
Add 5 µL loading buffer to concentrate and run for 00:45:00 at 90 Volts.
45m
Excise resultant band using sterile scapel, and visualised on blue light Led trans illuminator
Extract DNA from agarose Gel using QIAquick Gel Extraction Kit - QIAGEN under manufactuers standard protocol. Samples eluted in 30 µL buffer EB.
Quantification
Quantification
Gel Purified libraries are quantified using Invitrogen™ Qubit™ 1X dsDNA High Sensitivity (HS) quantification kit
Under manufacturers protocol and companion qubit fluorometer
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!
Sequencing on V2 or V3 Miseq
Sequencing on V2 or V3 Miseq
7m
7m
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 °C fridge. While reagent cartridge is thawing, perform steps 16-20
Prepare fresh 0.1 Molarity (M) NaOH.
To a 1.5 mL tube, add 10 µL of library and 10 µL of 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.
Allow the tubes to incubate at room temperature for 00:05:00 . Immediately add 980 µL of ice-cold Hyb to the library tube, and 990 µL Hyb to the PhiX tube.
Note: the resulting 20pM PhiX can be frozen and used for subequent runs.
5m
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
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 °C for 00:02:00 . Then cool immediately on ice.
2m
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
Load 600 ul of the final Libary/PhiX solution into well 17 on the reagent cartridge.
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.
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.
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.
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%
When run complete, perform post run wash as per manufacturers recommendations.