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Accurate quantification of the number of amplifiable library molecules loading into a flow cell is one of the most critical steps in the next-generation sequencing (NGS) workflow in obtaining high-quality read data with NGS technologies. Loading insufficient amount of library DNA will result in low cluster density and reduced sequencing yield. An overabundance of library DNA may increase cluster density and result in poor quality data. Standard methods of NGS library quantification, by electrophoresis or spectrophotometry, have low sensitivity, are non-specific for adapter-bound DNA and typically require a large amount of library sample for analysis. With its greater sensitivity and broad dynamic range, qPCR is therefore seen as the gold standard for NGS library quantification, as it accurately measures the number of molecules that can serve as templates during library and cluster amplification, even with very dilute libraries.
Meridian has developed the JetSeq™ Library Quantification Kit, an optimized, robust SYBR® Green based qPCR kit that provides accurate quantification of Illumina based NGS libraries. The JetSeq Library Quantification Kit contains pre-diluted standards to minimize pipetting errors, a pre-qualified P5 and P7 Illumina adaptor sequence primer mix to ensure reproducible and precise qPCR results and an optimized buffer for dilution of NGS library samples.
JetSeq Library Quantification Kit Performance - amplification curve.
qPCR amplification of each of the six supplied pre-diluted DNA standards (blue), from 10 pM to 100 aM and a tenfold serial dilution of the pre-diluted Illumina NGS library (red) are carried out with the supplied primer sets in triplicate.
JetSeq Library Quantification Kit Performance – melt curve.
Melt curve is performed to verify amplification of a single specific product for both standards and library even at the lowest concentrations.
JetSeq Library Quantification Kit Performance – standard curve.
From the standard curve a size adjusted library concentration is then determined. The amplification plots demonstrate a limit of detection of 100 aM.
JetSeq Library Quantification Kit lot‑to‑lot variation.
Three different lots of the JetSeq Library Quantification Kit were compared by generating an amplification plot using the standards from each kit in triplicate and averaged and running on the Mic Personal qPCR Cycler. The results illustrate that the JetSeq Library Quantification Kit delivers exceptional accuracy in library quantification.
Reagent |
500 x 20 μL |
JetSeq Primer Mix |
2 x 1.25 mL |
JetSeq FAST Hi-ROX Mix |
5 x 1 mL |
JetSeq Dilution Buffer |
5 x 5 mL |
DNA Standard 1 (10 pM) |
1 x 300 μL |
DNA Standard 2 (1 pM) |
1 x 300 μL |
DNA Standard 3 (100 fM) |
1 x 300 μL |
DNA Standard 4 (10 fM) |
1 x 300 μL |
DNA Standard 5 (1 fM) |
1 x 300 μL |
DNA Standard 6 (100 aM) |
1 x 300 μL |
On Dry Ice or Blue Ice.