qPCR general protocol for NGS sequencing

KAPA HiFi™ Library Amplification Kit Protocol

Library amplification protocol

Step 1: Preparation
- Thaw the primers required for PCR enrichment (see Table 1 for details) and a tube of KAPA HiFi HotStart Real-Time PCR Master Mix (2X), and fluorescent standards 1 - 4 at room temperature.
- Mix and briefly centrifuge the thawed KAPA HiFi HotStart Real-Time PCR Master Mix (2X), primer and fluorescent standards 1 - 4 for 5 seconds at 600 x g.
- Thaw and briefly centrifuge the adaptor-ligated, size-separated purified library DNA for 5 seconds at 600 x g.
- Pre-program the real-time thermal cycler using the recommended cycling protocol supplied in Table 1 for the specific set of library amplification PCR primers.

Step 2: Reaction Setup
Each plate must contain a set of fluorescent standards 1 - 4 (each loaded in triplicate) in addition to a single 50μL real-time PCR reaction for each library requiring amplification.
In order to maintain optimal library diversity it is necessary to add sufficient adaptor-ligated library DNA to each enrichment PCR reaction. The optimal cycle number is dependant on the volume and concentration of library material added to each 50 µL PCR reaction. High background fluorescence may result if >100 ng dsDNA template is added per 50μL real-time PCR reaction.
To each reaction add the following components changing tips after each pipetting step. Consult Table 1 for the suggested reaction setup for specific library preparation protocols.

2.1: Sample setup

• 25μL KAPA HiFi HotStart Real-Time PCR Master Mix (2X)
• Primer mix or each individual primer
• Purified adaptor-ligated library DNA
• Make up to 50μL with PCR-grade water

2.2: Fluorescent standard setup

• Add 50μL of each fluorescent standard in triplicate to wells of the real-time PCR plate
• Seal each reaction, mix gently and centrifuge for 5 seconds at 600 x g

Step 3: Cycling protocol
Refer to Table 1 for the thermal cycling protocol for specific library types.
* If conventional end-point PCR has previously been used successfully and the same amount and type of library is added to the KAPA HiFi HotStart Real-Time PCR reactions, then program the real-time thermocycler with the same number of cycles as previously used.
* It is important to ensure that data acquisition is performed at 72 ºC.

Step 4: Clean up PCR
After enrichment PCR, clean up each reaction using either Agencourt AMPure XP beads (Beckman Coulter Genomics part # A63881) or Qiagen MinElute PCR Purification Kit (Qiagen, part # 28004).

Step 5: Validate library
Initially, the raw data (i.e., not background subtracted) linear real-time amplification plots can be used as a built-in quality metric to validate the level of amplification of each amplified library.
* If the linear amplification profile of the library is significantly below fluorescent standard 1 at the end of qPCR cycling, then it is unlikely that there will be sufficient library material to sequence after PCR purification.
* If the linear amplification profile of the library is significantly above fluorescent standard 3 at the end of qPCR cycling, then the library has been over-amplified. This may lead to 1) amplification bias, 2) higher error rates, and/or 3) the presence of chimeric PCR products.
This data is also useful as a quality control metric for identifying inconsistencies during library preparation between multiple libraries.
NOTE: The amplification plots can also be used in real-time to select the optimal cycle without a pre-programmed termination cycle. To do this:
1. Program 30 cycles into the real-time thermocycler. 2. After starting the real-time thermocycler, wait until the desired fluorescence of the library is achieved before terminating the real-time reaction. NOTE: It is critical to terminate the reaction directly after data acquisition at 72ºC and before the tube ramps to 95ºC for the start of the next cycle. This will ensure that the enriched library DNA remains double-stranded forefficient downstream purification. To verify the size of the PCR enriched fragments, check the size distribution by performing gel electrophoresis.

5.1: Library Quantification
Accurate quantification of amplifiable library molecules is critical for the efficient use of next-generation sequencing platforms. Overestimation of library concentration results in lower cluster density after bridge PCR. Underestimation of library concentration results in too many clusters on the flow cell, which can lead to poor cluster resolution. Both scenarios result in suboptimal sequencing capacity. Accurate library quantification is equally important when pooling indexed libraries for multiplexed sequencing to ensure equal representation of each library. Integrate KAPA HiFi Real-Time PCR Library Amplification Kit with the appropriate KAPA Library Quantification Kit to accurately quantify the number of PCR-competent molecules. If libraries have been terminated between fluorescent standards 1– 3, a single 1:1,000 dilution of each library will be required for library quantification using the KAPA Library Quantification Kits.