Products >> Next Generation Sequencing (NGS) >> Library Amplification Reagents
.............................................................................................................................................................................................

KAPA Library Amplification Kits

Reduce amplification bias and improve sequencing coverage.

High fidelity PCR in NGS is used to selectively enrich library fragments carrying appropriate adaptor sequences and to amplify the amount of DNA prior to sequencing. During PCR enrichment, existing high fidelity DNA polymerases do not synthesize all library fragments with equal efficiency. This amplification bias exacerbates uneven sequence coverage.

KAPA HiFi Library Amplification Kits contain a novel DNA polymerase engineered for:

	Improved amplification of GC- and AT-rich genomic regions
	Reduced enzyme bias resulting in improved sequencing coverage
	Industry leading fidelity

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.

3GC content is known to be an important factor in NGS library amplification bias (Aird et al. Genome Biology 2011, 12:R18). DNA polymerases commonly used for library amplification display significant bias against GC- and AT-rich regions. Genomes with extreme average GC content are particularly susceptible to amplification bias. High-GC content elements such as 5’ UTRs, CpG islands and first exons, contained within the balanced human genome, are also vulnerable to amplification bias (Zhang L et al. PNAS 2004; 101:16855-16860).

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.

Percentage of the M. tuberculosis genome not represented in sequence data when using different PCR reagents for library amplification.

Indexed libraries were prepared from identical sheared M. tuberculosis (65% GC) gDNA using the illumina TruSeq™ DNA Sample Prep Kit and then amplified using the indicated polymerases before paired-end sequencing (2 x 75 bp). Libraries were quantified before and after amplification using the KAPA Library Quantification Kit to determine the number of doublings in each case. After filtering and aligning read pairs to reference sequences, 250 000 read pairs (~8.5x coverage) were randomly sampled for each genome.

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.

Percentage of the P. falciparum genome not represented in sequence data when using different PCR reagents for library amplification.

Equivalent indexed libraries were amplified (14 PCR cycles) using either KAPA HiFi Hot Start Ready mix or Phusion HF master mix, and then sequenced and compared to an unamplified control library. Average coverage depth for each library was 16.0x (unamplified control); 16.5x (KAPA HiFi); 18.8x (Phusion).

Library amplification can dramatically affect coverage uniformity.

The following Artemis screen captures depict examples of coverage bias in libraries amplified with either KAPA HiFi HotStart Master Mix or Phusion® HF Master Mix, compared to an unamplified control library. In short stretches of either high or low-GC content, the degree of coverage bias varies according to the method used to amplify the library.

Coverage depth and GC content across a ~7 kb region of the P. falciparum genome. Within this region of the genome there are 3 locations where high-AT sequences (>80%) lead to coverage bias (grey bars). In all three regions coverage depth drops significantly after amplification with Phusion® (blue), while the library amplified using KAPA HiFi (green) shows more uniform coverage depth which tracks that of the unamplified control library (black).

Coverage depth and GC content across a ~7 kb region of the B. pertussis genome. Within this region of the genome there are 4 distinct locations of high-GC sequence (>75%) that lead to sequence coverage bias (grey bars). in these regions the library amplified using Phusion® (blue) exhibits lower depth of coverage compared to the unamplified control. In contrast, the library amplified with KAPA HiFi (green) exhibits more even coverage depth, similar to the control library (black).

Superior accuracy for high fidelity library amplification.

Error rates of DNA polymerases and polymerase blends. The error rate of KAPA HiFi is calculated at 1 error in 3.54 x 106 bases covered (2.82 x 10-7). The error rate of KAPA HiFi is 100X lower than Taq polymerase, 40X lower than polymerase blends and 2X lower than Phusion®.

.............................................................................................................................................................................................