TITLE: Sample Preparation for Illumina MiSeq Dual Index Amplicon Sequencing
AUTHORS: Máté  Vass, Anna Székely

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Amplicon sequencing sample preparation for bacteria and microeukaryotes</div></div>

[STEPS]
?. Perform the first PCR (duplicates of each sample) using Illumina adaptor attached primers that target the gene of your choice. Here we present the protocol using the bacterial primers 341F and 805RN, and eukaryotic primers 574*f and 1132r. Bacterial primers (Herlemann et al., 2011):Illumina adapter-N4-341F:5’-ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCCTACGGGNGGCWGCAG-3’Illumina adapter-805R:5’-AGACGTGTGCTCTTCCGATCTGACTACHVGGGTATCTAATCC-3’Eukaryotic primers (Hugerth et al., 2014):Illumina adapter-4N-574*f:  5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCGGTAAYTCCAGCTCYV-3'Illumina adapter-1132r: 5'-AGACGTGTGCTCTTCCGATCTCCGTCAATTHCTTYAART-3'First PCR reactions  ABCDEF1Components
Working conc.
Final conc.
1 reaction (20 µl)
(N) reactions
&nbsp;
25xQ5 Reaction Buffer
5X
1X
4 µl
&nbsp;
&nbsp;
3Forward Primer10 µM
0.25 µM
0.5 µl&nbsp;
&nbsp;
4Reverse Primer10 µM
0.25 µM
0.5 µl&nbsp;
&nbsp;
5dNTPs
&nbsp;2 mM
200 µM
2 µl
&nbsp;
&nbsp;
6Q5 HF DNA polymerase
2 U/µl
0.02 U/µl
0.2 µl
&nbsp;
&nbsp;
7Template DNA
&nbsp;
&nbsp;
1 µl
&nbsp;
&nbsp;
8Nuclease-Free water
&nbsp;
&nbsp;
11.8 µl
&nbsp;
&nbsp;
9∑
&nbsp;
&nbsp;
20 µl
&nbsp;
&nbsp;
First PCR program for bacteria  ABC1STEP
TEMP.
TIME
2Initial Denaturation
98oC
30 seconds3&nbsp;
20 cycles
98oC
10 seconds
4Annealing temp.48°C*30 seconds572°C30 seconds/kb6Final elongation72oC
2 minutes
7Hold
6oC
∞
First PCR program for eukaryote  ABC1STEPTEMP.TIME2Initial Denaturation98°C1 minute320 cycles98°C10 seconds4Annealing temp.51°C30 seconds572°C1 minute/kb6Final elongation72°C2 minutes7Hold6°C∞
ABCDEF1Components
Working conc.
Final conc.
1 reaction (20 µl)
(N) reactions
&nbsp;
25xQ5 Reaction Buffer
5X
1X
4 µl
&nbsp;
&nbsp;
3Forward Primer10 µM
0.25 µM
0.5 µl&nbsp;
&nbsp;
4Reverse Primer10 µM
0.25 µM
0.5 µl&nbsp;
&nbsp;
5dNTPs
&nbsp;2 mM
200 µM
2 µl
&nbsp;
&nbsp;
6Q5 HF DNA polymerase
2 U/µl
0.02 U/µl
0.2 µl
&nbsp;
&nbsp;
7Template DNA
&nbsp;
&nbsp;
1 µl
&nbsp;
&nbsp;
8Nuclease-Free water
&nbsp;
&nbsp;
11.8 µl
&nbsp;
&nbsp;
9∑
&nbsp;
&nbsp;
20 µl
&nbsp;
&nbsp;
ABC1STEP
TEMP.
TIME
2Initial Denaturation
98oC
30 seconds3&nbsp;
20 cycles
98oC
10 seconds
4Annealing temp.48°C*30 seconds572°C30 seconds/kb6Final elongation72oC
2 minutes
7Hold
6oC
∞
ABC1STEPTEMP.TIME2Initial Denaturation98°C1 minute320 cycles98°C10 seconds4Annealing temp.51°C30 seconds572°C1 minute/kb6Final elongation72°C2 minutes7Hold6°C∞
?. Check PCR products with Agarose gel electrophoresis (1%) - optional
?. Pool PCR duplicate samples together Optional: run Agarose gel electrophoresis (1%)
?. Second PCR is conducted for attaching standard illumina handles and index primersMultiplex_fwdAATGATACGGCGACCACCGAGA{TCTACAC}-[i5 index] ACACTCTTTCCCTACACGACGMultiplex_revCAAGCAGAAGACGGCATACGAGAT-[i7 index]-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT(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)Second PCR reactions  ABCDEF1Components
Working conc.
Final conc.
1 reaction (20 µl)
(N) reactions
&nbsp;
25xQ5 Reaction Buffer
5X
1X
4 µl
&nbsp;
&nbsp;
3Forward index (i5, illu-N501-N508)
5 µM
0.25 µM
1 µl
&nbsp;
&nbsp;
4Reverse index (i7, illu-N701-N712)
5 µM
0.25 µM
1 µl
&nbsp;
&nbsp;
5dNTPs
&nbsp;2 mM
200 µM
2 µl
&nbsp;
&nbsp;
6Q5 HF DNA polymerase
2 U/µl
0.02 U/µl
0.2 µl
&nbsp;
&nbsp;
7Template from 1st PCR
&nbsp;
&nbsp;
2 µl
&nbsp;
&nbsp;
8Nuclease-Free water
&nbsp;
&nbsp;
9.8 µl
&nbsp;
&nbsp;
9∑
&nbsp;
&nbsp;
20 µl
&nbsp;
&nbsp;
Second PCR program for both bacteria and eukaryote  ABC1STEP
TEMP.
TIME
2Initial Denaturation
98oC
30 seconds
3&nbsp;
15 cycles
98oC
10 seconds
466°C30 seconds572°C30 seconds/kb6Final Elongation72oC
2 minutes
7Hold
6oC
∞
ABCDEF1Components
Working conc.
Final conc.
1 reaction (20 µl)
(N) reactions
&nbsp;
25xQ5 Reaction Buffer
5X
1X
4 µl
&nbsp;
&nbsp;
3Forward index (i5, illu-N501-N508)
5 µM
0.25 µM
1 µl
&nbsp;
&nbsp;
4Reverse index (i7, illu-N701-N712)
5 µM
0.25 µM
1 µl
&nbsp;
&nbsp;
5dNTPs
&nbsp;2 mM
200 µM
2 µl
&nbsp;
&nbsp;
6Q5 HF DNA polymerase
2 U/µl
0.02 U/µl
0.2 µl
&nbsp;
&nbsp;
7Template from 1st PCR
&nbsp;
&nbsp;
2 µl
&nbsp;
&nbsp;
8Nuclease-Free water
&nbsp;
&nbsp;
9.8 µl
&nbsp;
&nbsp;
9∑
&nbsp;
&nbsp;
20 µl
&nbsp;
&nbsp;
ABC1STEP
TEMP.
TIME
2Initial Denaturation
98oC
30 seconds
3&nbsp;
15 cycles
98oC
10 seconds
466°C30 seconds572°C30 seconds/kb6Final Elongation72oC
2 minutes
7Hold
6oC
∞
?. Check second PCR products with Agarose gel electrophoresis (1%)
?. Perform purification with magnetic beads (Agencourt AMPure) and run Agarose gel electrophoresis (1%)
?. Quantification: PicoGreen assay (Quant-iT PicoGreen dsDNA Assay Kit, Invitrogen)
?. 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-one tube with a mix of all the samples (bacteria and eukaryote separately) 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.
?. Requantify with PicoGreen before submitting to sequencing facility.