Accurate, reliable and reproducible qPCR-based method for quantifying DNA libraries prior to NGS on Illumina platforms.
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.
Melt curve is performed to verify amplification of a single specific product for both standards and library even at the lowest concentrations.
From the standard curve a size adjusted library concentration is then determined. The amplification plots demonstrate a limit of detection of 100 aM.
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.
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.
| Presentation | BIO-65027 62.5 µL (2,500 units) |
| – JetSeq Primer Mix | 2 x 1.25 mL |
| – JetSeq FAST Lo-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 |
| Appearance | Clear, colorless solutions |
| Hot start | Antibody mediated |
| Application | Quantification of individual libraries or library pools prior to cluster amplification, Quantification of libraries prior to pooling for multiplexed sequencing, Quality control, optimization and troubleshooting of library construction processes or workflows |
| Sample type | Sequencing by synthesis NGS library DNA |
| Presentation | 18 vial |
| Storage | -20 °C, avoid exposure of the SYBR™ and ROX™ to light |
| Mix stability | See outer label |
| Specific Activity | 2x |
| Consistency | ±0.5 Ct variance between test and reference sample |
| DNA Contamination | None detected in PCR amplification with traces overlay with the negative control on E. coli and mouse genomic DNA specific targets. |
| DNase Contamination | No detectable degradation |
Cat. No. Size
BIO-65027 500 Reactions
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