Feb 04, 2025

Public workspaceillumina MiSeq Dual Index Amplicon Sequencing Sample Preparation Eukarytic 18S rRNA gene

 Forked from illumina MiSeq Dual Index Amplicon Sequencing Sample Preparation Bacterial 16S rRNA gene
This protocol is a draft, published without a DOI.
  • Alexander Eiler1,
  • Eivind Stensrud2
  • 1University of Oslo;
  • 2eDNA solutions AB
  • AQUA at UiO
  • eDNAsolutions
Alexander Eiler
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Protocol Citation: Alexander Eiler, Eivind Stensrud 2025. illumina MiSeq Dual Index Amplicon Sequencing Sample Preparation Eukarytic 18S rRNA gene. protocols.io https://protocols.io/view/illumina-miseq-dual-index-amplicon-sequencing-samp-dyyj7xun
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 04, 2025
Last Modified: February 04, 2025
Protocol Integer ID: 119531
Keywords: rrna gene preparation, pcr products for amplicon, pcr product, pcr, amplicon, rrna gene, sequencing, sample preparation
Disclaimer
Use at your own risk
Abstract
Preparation of PCR products for amplicon sequencing
Guidelines
We have a common stock of barcoded primers stored in the -80C freezer number 3. The primers are in an orange box, which box can be found in the position indicated on the following picture:


In the box from 1 to 20 are the forward primers and from 37 to 56 are the reverse primers. The last line has extras from some primers. If you run out of a barcode, check those first before ordering. Check primer ordering file on common server for primer ordering. 
Sequence of barcodes to fill in the SampleSheet file for sequencing:
Index 2 (i5)Index 2 (i5) SequenceIndex 1 (i7)Index 1 (i7) Sequence
Illu_N501FTAGATCGCIllu_N701RTCGCCTTA
Illu_N502FCTCTCTATIllu_N702RCTAGTACG
Illu_N503FTATCCTCTIllu_N703RTTCTGCCT
Illu_N504FAGAGTAGAIllu_N704RGCTCAGGA
Illu_N505FGTAAGGAGIllu_N705RAGGAGTCC
Illu_N506FACTGCATAIllu_N706RCATGCCTA
Illu_N507FAAGGAGTAIllu_N707RGTAGAGAG
Illu_N508FCTAAGCCTIllu_N708RCCTCTCTG
Illu_N521FCTTGCTTTIllu_N709RAGCGTAGC
Illu_N522FGGCTTCAAIllu_N710RCAGCCTCG
Illu_N523FAATCGGCAIllu_N711RTGCCTCTT
Illu_N524FGGTTCAAAIllu_N712RTCCTCTAC
Illu_N525FACTTCGACIllu_N733RGGTATAAG
Illu_N526FTGACTTGCIllu_N735RCAGCTAGA
Illu_N527FTAGGACCTIllu_N736RCCATAGCA
Illu_N528FGGAGACTTIllu_N738RGGTATAGC
Illu_N529FAGGTTACGIllu_N739RGGTTATGC
Illu_N530FAATTCGCTIllu_N740RTAGGCAAG
Illu_N531FTCAGCTAAIllu_N741RTTGTCCAT
Illu_N532FGCGATATGIllu_N743RTCTAGGCA
Note: the sequence of the reverse barcodes to fill in the SampleSheet is the reverse complement of their sequence of in the primers.
The 1st part of the protocol is performed in the pre-PCR room. 
The 2nd part in the post-PCR room.
Never bring back PCR products to the pre-PCR room.
Materials
MATERIALS
ReagentQ5 High-Fidelity DNA Polymerase - 100 unitsNew England BiolabsCatalog #M0491S
Protocol materials
ReagentQ5 High-Fidelity DNA Polymerase - 500 unitsNew England BiolabsCatalog #M0491L
Troubleshooting
Before start
Put pipettes and tips in the UV chamber for 10 mins.
Clean bench with MQ and EtOH.
Detailed protocol
Perform the first PCR (triplicates/duplicates of each sample) using Illumina adaptor attached primers targeting your chosen gene. Here, we present the protocol using the Eukaryotic primers TAR euk 454F and V4 18S Next.Rev. For the forward primer cite: Stoeck et al., 2010 and for the reverse primer cite based on: Piredda et al., 2017.
Illumina adapter-N4-TAR euk 454F:
5’-ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCCAGCASCYGCGGTAATTCC-3’
Illumina adapter-V4 18S Next.Rev:
5’-AGACGTGTGCTCTTCCGATCTCCAGCASCYGCGGTAATTCC-3’
First PCR reactions
ABCDEF
Components Working conc. Final conc. 1 reaction (20 µl) (N) reactions  
5xQ5 Reaction Buffer 5X 1X 4 µl    
Forward Primer (illu-ada-TAR euk 454F) 10 µM 0.25 µM 0,5 µl    
Reverse Primer (illu-ada-V4 18S Next.Rev) 10 µM 0.25 µM 0,5 µl    
dNTPs  2 mM 200 µM 2 µl    
Q5 HF DNA polymerase 2 U/µl 0.02 U/µl 0.2 µl    
Template DNA     1 µl    
Nuclease-Free water     11.8 µl    
∑     20 µl    
First PCR program
ABC
STEP TEMP. TIME
Initial Denaturation 98oC 3 minutes*
  25 cycles 98oC 10 seconds
60oC** 30 seconds
72oC 30 seconds/kb
Final Extension 72oC 5 minutes
Hold 6oC ∞
* In the latest protocols this was reduced to 30 seconds but in order to have complete denaturation of long high GC genome fragments, we increased it to 3 minutes, which is in accordance with the manufacturers recommendation.
** This may be adjusted to lower temperatures potentially leading to unspecific amplification. If you use this profile cite: addressing PCR biases or effect of annealing temperature
ReagentQ5 High-Fidelity DNA Polymerase - 500 unitsNew England BiolabsCatalog #M0491L
Check PCR products with Agarose gel electrophoresis (1%) - optional
Pool PCR duplicate samples together and perform purification with magnetic beads. The magnetic beads employed were Cytiva-Sera-Magand the specified protocol (https://dx.doi.org/10.17504/protocols.io.n2bvj393wlk5/v1 ) was followed- Optional: run Agarose gel electrophoresis (1%)
  1. A second PCR is conducted for attaching standard illumina handles and index primers
Multiplex_fwd
AATGATACGGCGACCACCGAGA{TCTACAC}-[i5 index] ACACTCTTTCCCTACACGACG
Multiplex_rev
CAAGCAGAAGACGGCATACGAGAT-[i7 index]-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
(We have in total 20 different forward index/barcode primers and 20 different reverse index/barcode primers. By combining both primers (20X20), it can possible to generate 400 tags in one final pool for sequencing. To find the common stock of the barcoded second step primers check the guidelines of this protocol)
Table 2: Second PCR reactions
Components Working conc. Final conc. 1 reaction (20 µl) (N) reactions  
5xQ5 Reaction Buffer 5X 1X 4 µl    
Forward index (i5, illu-N501-N508) 5 µM 0.25 µM 1 µl    
Reverse index (i7, illu-N701-N712) 5 µM 0.25 µM 1 µl    
dNTPs  2 mM 200 µM 2 µl    
Q5 HF DNA polymerase 2 U/µl 0.02 U/µl 0.2 µl    
Template from 1st PCR     2 µl    
Nuclease-Free water     9.8 µl    
∑     20 µl    
Second PCR program
STEP TEMP. TIME
Initial Denaturation 98oC 30 seconds
  15 cycles 98oC 10 seconds
66oC 30 seconds
72oC 30 seconds/kb
Final Extension 72oC 2 minutes
Hold 6oC ∞
Check second PCR products with Agarose gel electrophoresis (1%)
Duration00:01:00
Perform purification with magnetic beads. The magnetic beads employed were Cytiva-Sera-Magand the specified protocol (https://dx.doi.org/10.17504/protocols.io.n2bvj393wlk5/v1 ) was followed- Optional: run Agarose gel electrophoresis (1%)
Quantification: using gel analyzer program or PicoGreen assay
Calculate PCR samples concentration and volume before pooling
Pool the PCR samples in equal DNA amount (ng) or for unequal length amplicons, in equal molecule amount (mol).
You will get one tube with a mix of all the samples in it.
To calculate the volume of each sample to be pooled (DNA amount mixing):
  • Use the lowest concentration sample to define the minimum amount of DNA (ng) that you have available from a single sample: the DNA concentration (ng/μL) of the lowest concentration sample multiplied with its volume (μL). This will be your target DNA amount for each sample.
  • Calculate how many μLs of each sample you need to achieve the target DNA amount: divide the target DNA amount with the concentration of each sample.
  • Pipette into one tube the calculated volume of each sample. Aim to use the same pipette for all samples (dilute or pipette multiple times) to avoid pipette calibration errors.
Gel purify the pool and requantify with PicoGreen before submitting to sequencing facility.