NGS library amplification and quantification solutions

To ensure high sequencing data quality and yields, both library amplification (when appropriate) and library quantification must be performed with high accuracy, low bias, and high efficiency. KAPA library amplification and NGS library quantification enzyme mixes contain enzymes purposefully engineered for efficiency for a wide range of GC contents and fragment sizes. 

KAPA HiFi DNA Polymerase, the enzyme contained in KAPA EvoAmp ReadyMixes and updated KAPA Library Amplification Kits, offers ultra-high fidelity to mitigate against the introduction of errors during NGS library amplification.¹ KAPA Library Quantification Kits contain the engineered KAPA SYBR FAST enzyme, engineered for robust and reproducible SYBR Green I-based qPCR.²

Most NGS workflows include at least one library amplification step; this includes DNA workflows (except PCR-free WGS), most RNA-seq workflows, and most targeted NGS workflows. The roles of library amplification in NGS library prep are:

1. Generate sufficient material for the next step in the workflow; for example, to increase the ability to detect low-abundance cDNAs or to increase sample mass prior to target enrichment 
2. Add NGS index sequences when truncated universal adapters are used to convert DNA or cDNA fragments to library molecules (full-length adapters contain indexes, and may be used when PCR-free workflows are desired)

Thus, accurate and low-bias library amplification is essential to ensure that the final data represents the original sample, especially when the input DNA consists of low amounts or poor-quality nucleic acids such as ctDNA or FFPET DNA, which can require more rounds of amplification.

Quantification of NGS library quantification may be performed at multiple stages in a workflow. For example,

1. Quantification of indexed libraries prior to pooling for target enrichment (multiplexed capture) ensures that each sample is represented equally in the capture reaction.  
2. Quantification of post-capture libraries or of un-enriched libraries prior to sequencing helps to ensure optimal cluster generation on the instrument, which is important for both sequencing efficiency and data quality.

qPCR-based methods, such as the KAPA Library Quantification Kit, are more accurate at quantifying sequenceable library molecules than other methods, such as fluorometer or electrophoretic methods, as they only recognize sequenceable library fragments; the other methods will also include incomplete library fragments in their output.

PCR with traditional DNA polymerases is known to introduce bias, thereby compromising NGS data.³ Roche's KAPA library amplification solutions contain the engineered KAPA HiFi DNA Polymerase, valued in NGS library amplification due to its ability to amplify complex DNA populations with ultra-high fidelity, high efficiency, and low bias.⁴

• KAPA EvoAmp ReadyMix, KAPA HiFi HotStart ReadyMix, and all KAPA Library Amplification Kits offer high amplification efficiency across a wide range of fragment lengths, GC contents, and sequencing motifs, thereby reducing the amount of PCR cycles needed to achieve high success rates with routine and challenging samples⁵
• Highly-efficient, low-bias amplification preserves NGS library complexity to ensure sequencing results that are representative of the original sample
• Low bias amplification is essential for achieving high coverage uniformity, which reduces dropout and duplication rates and contributes to improved sequencing economy⁶