TITLE: illumina MiSeq Dual Index Amplicon Sequencing Sample Preparation Bacterial 16S rRNA gene
AUTHORS: Alexander Eiler, Omneya Osman, Anna Szekely

[DESCRIPTION]
Preparation of PCR products for amplicon sequencing

[BEFORE_START]
Put pipettes and tips in the UV chamber for 10 mins.
Clean bench with MQ and EtOH.

[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) Sequence Index 1 (i7) Index 1 (i7) Sequence   Illu_N501F TAGATCGC Illu_N701R TCGCCTTA   Illu_N502F CTCTCTAT Illu_N702R CTAGTACG   Illu_N503F TATCCTCT Illu_N703R TTCTGCCT   Illu_N504F AGAGTAGA Illu_N704R GCTCAGGA   Illu_N505F GTAAGGAG Illu_N705R AGGAGTCC   Illu_N506F ACTGCATA Illu_N706R CATGCCTA   Illu_N507F AAGGAGTA Illu_N707R GTAGAGAG   Illu_N508F CTAAGCCT Illu_N708R CCTCTCTG   Illu_N521F CTTGCTTT Illu_N709R AGCGTAGC   Illu_N522F GGCTTCAA Illu_N710R CAGCCTCG   Illu_N523F AATCGGCA Illu_N711R TGCCTCTT   Illu_N524F GGTTCAAA Illu_N712R TCCTCTAC   Illu_N525F ACTTCGAC Illu_N733R GGTATAAG   Illu_N526F TGACTTGC Illu_N735R CAGCTAGA   Illu_N527F TAGGACCT Illu_N736R CCATAGCA   Illu_N528F GGAGACTT Illu_N738R GGTATAGC   Illu_N529F AGGTTACG Illu_N739R GGTTATGC   Illu_N530F AATTCGCT Illu_N740R TAGGCAAG   Illu_N531F TCAGCTAA Illu_N741R TTGTCCAT   Illu_N532F GCGATATG Illu_N743R TCTAGGCA   
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.

[STEPS]
1. Perform the first PCR (triplicates/duplicates of each sample) using Illumina adaptor attached primers that target the gene of your choice. Here we present the protocol using the Bacteria primers 341F and 805RN. For the forward primer cite: Herlemann et al., 2011 and for the reverse primer cite based on: Apprill et al., 2015 but the one here is not exactly as the one in that paper.
 
Illumina adapter-N4-341F:
5’-ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCCTACGGGNGGCWGCAG-3’
Illumina adapter-805NR:
5’-AGACGTGTGCTCTTCCGATCTGACTACNVGGGTATCTAATCC-3’
 
First PCR reactions
 
 
 
 
First PCR program
 
 
* 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.
** First temperature ever used was 55C. Then it changed to 62C. We decided on 48 based on suggestion by Anna Szekely. 48C gets unbiased product amounts of non-mismatch and 3-mismatch isolates. If you use this profile cite: addressing PCR biases or effect of annealing temperature
 
2. Check PCR products with Agarose gel electrophoresis (1%) - optional
3. Pool PCR duplicate samples together and perform purification with magnetic beads (Agencourt AMPure or similar)- Optional: run Agarose gel electrophoresis (1%)
4. 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
 
 
 
Second PCR program
 
 
 
5. Check second PCR products with Agarose gel electrophoresis (1%)
1 min
6. Perform purification with magnetic beads (Agencourt AMPure) and (optional) run Agarose gel electrophoresis (1%)
7. Quantification: using gel analyzer program or PicoGreen assay
8. Calculate PCR samples concentration and volume before pooling
9. 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.
 
10. Gel purify the pool and requantify with PicoGreen before submitting to sequencing facility.