For Research Use Only. Not for use in diagnostic procedures.
The KAPA NGS FFPE DNA QC Kit offers a reliable qPCR-based qualification and recommended input assessment of human FFPE DNA samples prior to NGS library construction. Each kit contains an optimized high performing qPCR Master Mix, a DNA QC standard and primers in premixes formats. The primers target the human Long Interspersed Nuclear Elements (LINE) offering increased sensitivity and minimizing the amount of sample needed for QC.
Sample quality is assessed by relative quantification across a short and a longer amplicon, compared to the DNA QC standard. The calculated normalized Quality score (Q-score) is used to calculate the required input amount of the sample, which offers the greatest probability for high quality library preparation. Q-scores generated with the kit may be used to predict the outcome of library preparation and optimize library construction parameters for FFPE DNA samples.
The KAPA FFPE DNA QC Kit is validated in the KAPA HyperPETE Tissue DNA Workflow but may be theoretically compatible with the KAPA HyperCap FFPET DNA Workflow.
The KAPA NGS FFPE QC Kit assesses sample quality by comparing the amplification of a larger (191-bp) amplicon and a smaller (66 bp) amplicon in LightCycler® qPCR assays using primers targeting the human Long Interspersed Nuclear Elements (LINE). Targeting LINE elements allows for a stronger signal from very low quantities of input FFPET DNA, resulting in a more sensitive result and minimal use of sample for QC testing. This allows for a reliable assessment of quality, which is averaged across the genome, as opposed to a single-copy housekeeping gene. In addition qPCR assays offer much higher sensitivity and accuracy in assessing sample quality than spectrophotometric, and electrophoretic methods.
Comparing the relative amplification of these two qPCR assays reveals the sample’s Q-score, which can be compared to the Q-score to a QC PCR DNA Standard of known quantity as an indicator of the sample’s quality. The amount of input DNA for lower quality samples can then be appropriately adjusted to increase the likelihood of library prep success.
Quality adjusted input (Q-input) dramatically improves sequencing coverage metrics for Low Quality (LQ), Medium Quality (MQ) and even High Quality (HQ) FFPE DNA samples in the KAPA HyperPETE Tissue DNA Workflow.
Figure 1. Using quality adjusted input by the KAPA NGS FFPE DNA QC Kit, the sequencing coverage metrics for the KAPA HyperPETE Pan Cancer and Hot Spot Panels dramatically improve, compared to non-adjusted input of 10 ng. The normalized Q-score for the low quality (LQ) FFPET DNA samples ranged from 0.042 to 0.2. The quality adjusted (Q-adjusted) input ranged from 60 to 248 ng in the KAPA HyperPETE Tissue DNA Workflow.
Figure 2. Quality adjusted input (Q-input) dramatically improved the % of panel exons covered by 500x or greater with the KAPA HyperPETE Pan Cancer Panel in the KAPA HyperPETE Tissue DNA Workflow. Compared to the coverage from 10 ng. Low Quality samples (LQ) show the most dramatic improvement. However, Medium Quality (MQ) and even High Quality (HQ) samples benefit as well by using Q-input as calculated by the KAPA NGS FFPE DNA QC Kit.