6.5. Second PCR with Indexed Primers

This step adds a unique UD dual index combination to each Anchored PCR Product generated in the previous PCR with the Anchor Primers step as well as universal flanking P5 and P7 sequences needed for cluster formation on the Illumina NGS flow cell (Outline).

The Index PCR Plate provided in the kit contains a complete set of 96 unique combinations of Forward and Reverse DNA/RNA UD index primers in each well. The primers have been dried onto the bottom of each well and will be dissolved when the PCR reaction mix with the sample is added. If you are processing fewer than 96 samples, you can use scissors to cut the desired number of wells from the index plate (e.g., cut 2 columns to run 4 samples in triplicate), then store the rest of the plate for later use. See Appendix D. List of Barcodes in the 96-well Plate for the sequences of each Forward and Reverse Index combination.

Arrangement of the index pairs for 96 samples

The protocol below specifies the volumes of reagents and Master Mix for each sample which can be 1 to 8 Pooled samples and positive control sample. In order to avoid low complexity reads in index NGS reads, we recommend to amplify all samples in triplicates using 3 different indexes.

1. Prepare the PCR Master Mix, following the formulation below for one sample, and increase proportionally if you run more samples:
    
   

   PCR Master Mix Component	Volume per sample, µl
   PCR Buffer, 5X	30
   dNTP Mix	3
   Water	115.5
   DNA Polymerase, 100X	1.5
   Total	150

2. Gently vortex the PCR Master Mix, and spin down briefly to collect droplets. Transfer 5 µl of Anchored PCR products after the first PCR to the test tube with a PCR Master Mix.
    
   

   Component	Volume per sample, µl
   Anchored PCR Product (from the step above)	5
   Index PCR Master Mix (above)	150
   Total	155

3. Mix the test tube contents by pipetting, and spin down to collect droplets. Remove the plate seal from the Index PCR Plate (provided in the kit), and aliquot 50 μl of the Anchored PCR product in PCR Master Mix into three different wells of the 96-well Index PCR Plate (or cut-off portion of the plate). We recommend that you record the Sample/replicate name and well number (e.g., Sample 1 in wells A1, A2, and A3) for all the samples. This will help minimize mistakes in the NGS deconvolution step.

4. Seal the plate (or portion of the plate) with new adhesive film, and spin it down to collect droplets. Load the plate in the thermal cycler, and run the following program:
    
   

   Temperature	Time	Cycles
   98°C	30 sec	1
   98°C	20 sec	8
   65°C	10 sec
   72°C	30 sec
   72°C	30 sec	1
   4°C	∞	1

!Stopping Point in procedure: PCR products may be stored at 4°C for a week.