Product introduction

1. Product application

　　The high efficiency, high fidelity, low preference amplification libraries before sequencing, mainly include:

(1) Complete genome shotgun sequencing

(2) Target sequencing (pre capture/post amplification)

(3) Amplifier sequencing

(4) ChIP sequencing

(5) RNA sequencing

2. Technical advantages

(1) To improve the amplification efficiency of high GC or high AT regions in the genome;

(2) reduce the amplification bias to improve the sequencing coverage;

(3) With industry-leading industrial-grade fidelity.

3. Excellent performance

(1) The influence of high GC content on the sequencing reading depth of libraries amplified with common proof-reading (B-family) amplifiers

(2) According to the different amplification enzymes used in library amplification, the libraries with low GC content would produce different amplification bias

Above: Libraries prepared with the same physically interrupted GDNA of P.falciparum(Plasmodium falciparum, AT 65%) were amplified using the PCR reagents (Kapahifi, Phusion®) shown in the figure, respectively, and then compared with equivalent libraries that were not amplified. The frequencies of different GC contents observed by sequencing READ under different conditions are shown in the curve in the figure (black = no amplification; Green = Kapa Hifi Hot Start Mastermix; Blue =Phusion® HFMasterMix). The expected frequency distribution of Reads is the gray shaded area in the figure. The curves of the unamplified library basically coincide with the outer edge of the desired frequency distribution region. The amplified library using Kapahifi showed minimal bias, while the amplified library using Phusion® showed large bias in terms of high GC content compared to the amplified library with low GC content. The average coverage depth of each library was 16.0x (as the unamplified sample of the control). 16.5 X (KAPA HiFi); 18.8 X (Phusion ®). Data provided by Dr.Michael A. Quuail, The Wellcome Trust Sanger Institute.

(3) Library amplification will greatly affect the consistency of sequencing coverage

　　The following screenshot in Artemis depicts the coverage bias after library amplification using Kapa Hifi Hot-started Master Mix and Phusion® HF Master Mix, respectively, compared to the control without library amplification. After different amplification pathways, no matter the fragment with high GC content or low GC content, the degree of coverage deviation is also different.

　　Above: Coverage depth and GC content of a 7kb fragment in the genome of P.falciparum

　　In this genome sequence, AT three locations, > content was 80%, resulting in coverage bias (areas highlighted in gray). In these three high-AT areas, the depth of coverage dropped sharply after library amplification using Phusion ®(blue); However, after library amplification using Kapa Hifi (green), the coverage depth of the three regions was consistent with that of other regions of the genome, and the coverage depth of the three regions was basically identical with that of the control group without amplification.

　Above: Coverage depth and GC content of a 7kb fragment in the B.pertussis(B.pertussis) genome

　　In this genome segment, four GC sites contained 75% >, resulting in coverage bias (areas highlighted in gray). In these four areas with high GC content, the depth of coverage decreased sharply when the library was amplified using Phusion ®(blue) compared to the control without amplification. After library amplification using Kapa Hifi (green), the coverage depth of the four regions was relatively uniform and consistent, which was similar to the results of the control group (black) without amplification.

(4) High accuracy of amplification of high-fidelity library

　　The error rate/mismatch rate of the Kapa Hifi amplified pair was only one error per 3.54×106 bases covered (2.82×10-7), which was 100 times lower than that of the Taq amplified pair, 40 times lower than that of the Taq-based hybrid, and two times lower than that of Phusion®.

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