Accurate quantification of amplifiable library molecules is critical for the efficient use of the Ion Torrent next generation sequencing (NGS) platform—overestimation results in too few DNA-bearing beads, while underestimation leads to multiple template molecules per bead during emPCR. Accurate library quantification is equally important when pooling indexed libraries for multiplexed sequencing to ensure equal representation of each library. qPCR is inherently well-suited to NGS library quantification, as it overcomes many of the difficulties posed by alternative approaches, for example it:
• quantifies only the PCR-amplifiable library molecules that are relevant to optimizing emPCR,
• is exceptionally sensitive and accurate across an extremely broad dynamic range, and
• is amenable to high sample throughput and automated liquid handling. The extreme sensitivity of qPCR enables accurate quantification of very dilute libraries. This minimizes the need for PCR amplification of libraries and the associated problematic biases.
KAPA Library Quantification Kits are comprised of DNA Standards (six 10-fold dilutions) and Primer Premix (10X), paired with KAPA SYBR® FAST qPCR Kits to accurately quantify the number of amplifiable molecules in an NGS library. The 153 bp KAPA Ion Torrent DNA Standard consists of a linear DNA fragment flanked by qPCR primer binding sites. Quantification is achieved by inference from a standard curve generated using the six DNA Standards.
KAPA Library Quantification Kits are vigorously tested to ensure minimal lot-to-lot variation. The kit contains the novel KAPA SYBR FAST DNA Polymerase, engineered through a process of directed evolution for highperformance SYBR Green I-based qPCR. The ability of the engineered polymerase to amplify diverse DNA fragments with similar efficiency enables the use of a universal standard for the reliable quantification of libraries with an average fragment length of up to 1kb, irrespective of library type or GC content. KAPA Ion Torrent Library Quantification Kits are suited for the quantification of libraries constructed with Ion Torrent adapters containing the following qPCR primer sequences:
Primer IT A: 5′-CCA TCT CAT CCC TGC GTG TC – 3′
Primer IT trP1: 5′-CCT CTC TAT GGG CAG TCG GTG AT-3′