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 Kits have been designed to address PCR-induced bias. Kits contain the novel KAPA HiFi DNA Polymerase, engineered for high fidelity and processivity and capable of balanced amplification of complex library DNA. Kits are supplied as a ready-to-use master mix (2X) containing all components for PCR, except primers and template.
KAPA HiFi HotStart DNA Polymerase is an antibody-based hot start formulation of KAPA HiFi DNA Polymerase, a novel B-family DNA polymerase exhibiting industry-leading performance in comparison with other high-fidelity (B-family) DNA polymerases and polymerase blends. KAPA HiFi DNA Polymerase was engineered for increased affinity to DNA, without the need for accessory protein domains. The intrinsic high processivity of the enzyme results in significant improvements in yield, sensitivity, speed, target length and the ability to amplify difficult amplicons. These enhancements result in lower amplification bias which leads to more uniform sequence coverage. In the HotStart formulation, a proprietary antibody inactivates the polymerase until the first denaturation step. This eliminates spurious amplification products resulting from non-specific priming events during reaction setup and initiation, and increases overall reaction efficiency.
KAPA HiFi HotStart DNA Polymerase has 5’➞3’ polymerase and 3➞5’ exonuclease (proofreading) activities, but no 5’➞3’ exonuclease activity. The strong 3’➞5’ exonuclease activity results in superior accuracy during DNA amplification. KAPA HiFi HotStart DNA Polymerase boasts the lowest error rate of all B-family DNA polymerases (1 error per 2.8 x 107 nucleotides incorporated). This fidelity is approximately 100X higher than that of wild-type Taq and up to 10X higher than that of other B-family DNA polymerases and polymerase blends. DNA fragments generated with KAPA HiFi HotStart ReadyMix may be used for routine downstream analyses or applications, including restriction enzyme digestion and sequencing. PCR products generated with KAPA HiFi HotStart ReadyMix are blunt-ended, but may be 3’-dA-tailed for cloning into TA cloning vectors.
Effect of high-GC content on coverage depth for libraries amplified using common proof-reading (B-family) polymerases
Indexed Illumina TruSeqTM libraries prepared from identical sheared M. tuberculosis (65% GC) gDNA were amplified using the indicated PCR reagents, and compared to an equivalent unamplified library by paired-end sequencing (2 x 75 bp). After filtering and aligning read pairs to reference sequences, 250 000 read pairs were randomly sampled for each genome, and scatter plots of mean sequence coverage depth vs. GC content were generated by analyzing 250 bp windows. GC-rich M. tuberculosis sequences were under-represented following library amplification using either Phusion® HF Master Mix or Illumina TruSeqTM PCR Master Mix. In contrast, library amplification with KAPA HiFi HotStart Master Mix resulted in coverage distribution across the range of GC-content that is almost indistinguishable from that of the unamplified control.
Low GC-content libraries result in variable bias depending on the polymerase used for amplification
Libraries prepared from identical sheared P. falciparum (19% GC) gDNA were amplified using the indicated PCR reagents, and compared to an equivalent unamplified library. Observed frequencies of GC-content for reads are plotted for each condition tested (black = unamplified; green = KAPA HiFi HotStart Master Mix; blue = Phusion® HF Master Mix). The expected frequency distribution of reads is indicated by the grey shaded area. The unamplified library tracked the expected frequency distribution. Amplification with KAPA HiFi showed minimal bias while amplification with Phusion® resulted in a dramatic bias against reads with low GC-content. Average coverage depth for each library was 16.0x (unamplified control); 16.5x (KAPA HiFi); 18.8x (Phusion®). Data courtesy of Dr. Michael A. Quail, The Wellcome Trust Sanger Institute.
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