PCR general protocol for NGS sequencing
High fidelity PCR is used to selectively enrich library fragments carrying appropriate adaptor sequences and to amplify the amount of DNA prior to sequencing. During PCR enrichment, the polymerase does not synthesize all library fragments with equal efficiency. This amplification bias exacerbates uneven sequence coverage.
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 Ready Mix (2X) at room temperature.
- Briefly centrifuge the thawed KAPA HiFi HotStart Ready Mix (2X) and primer tubes 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 thermal cycler using the recommended cycling protocol supplied in Table 1 for the specific type of Illumina library.
Step 2: Reaction Setup
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 uL PCR reaction. Titration PCR may be performed to optimize the yield prior to performing the preparative enrichment PCR reaction/s.
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.
- 25μL KAPA HiFi HotStart Ready Mix (2X)
- Primer mix or each individual primer
- Purified adaptor-ligated library DNA
- Make up to 50μL with PCR-grade water
- Seal each reaction, mix gently and centrifuge for 5 seconds at 600 xg
Step 3: Cycling protocol
Refer to Table 1 for the thermal cycling protocol for specific library types.
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
* To verify the size of the PCR enriched fragments, check the size distribution by performing gel electrophoresis.
* Use the appropriate KAPA Library Quantification Kit to accurately quantify the number of PCR-competent molecules. Accurate quantification of amplifiable library molecules is critical for the efficient use of the Illumina 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.