The Archer® PreSeq™ DNA QC Assay is a quantitative PCR (qPCR)-based method to determine the quality of the DNA in your sample prior to targeted library preparation using the Archer VariantPlex™ system. PreSeq addresses the importance of measuring the integrity of amplifiable DNA in your sample and is predictive of sample sequencing success.
The PreSeq DNA QC Assay can be used to pre-screen FFPE samples to determine DNA quality prior to NGS library preparation for sequencing. Using the PreSeq Assay in the early stage of library prep can save you time and money, maximizing the success rate of the assay by screening for samples that will yield quality libraries for sequencing and analysis.For Research Use Only. Not for use in diagnostic procedures.
The Archer DNA QC Assay is a qPCR-based assay used to evaluate the quantity of amplifiable DNA in a sample in relation to a known assay standard. This SYBR®-based assay amplifies a 100bp genomic DNA sequence in the sample and the assay standard in two separate reactions. A comparison of the two resultant quantification cycle (Cq) values results in a ΔCq, or DNA QC score. The DNA QC score holds predictive value for library yield and can be used to gauge the amount of input material required for successful VariantPlex library preparation.
Confidence in NGS data stems from knowledge that the information generated is representative of the sample analyzed. In order to ensure that the sample is accurately represented, data must be gathered prior to library preparation and must have aligning metrics post-sequencing. The Archer PreSeq DNA QC assay enables the user to gather critical information about the sample prior to library preparation that can be tied to sequencing data presented in Archer Analysis. The number of amplifiable genomes in the input is correlated to mean DNA start sites per gene-specific primer 2 (GSP2), a quality metric in Archer Analysis. The DNA QC score is indicative of sample complexity, and the mean DNA start sites per GSP2 is indicative of library complexity. An ideal QC assay aligns input and library complexity, which increases confidence that data represents the sample.
Statistical confidence in variant calling is reliant upon the number of unique reads covering the target region. As allele frequency drops due to tumor cellularity or heterogeneity of the biopsy, increasing amounts of unique reads are needed to confidently make a call. The reproducible identification of low allelic frequencies requires the interrogation of hundreds or potentially thousand of unique input molecules. Unfortunately, damage to input DNA molecules has the effect of decreasing the number of sequenceable input molecules contained in a given mass of DNA. Thus, although 10ng of DNA theoretically contains ~3,000 copies of every genomic locus, in reality, only a small fraction of those molecules may be available for library preparation. Therefore, mass alone does not help predict sequencing success or library quality.
Since the DNA QC score generated by PreSeq is dependent on DNA quality, it can be used to estimate the number of amplifiable genomes present in a sample. Amplifiable genomes are predictive of the assays sensitivity for variant and CNV detection. This information can be used to optimize input based on tumor cellularity, sample quality and sensitivity requirements. Samples that are expected to fail to generate library or fail to meet the labs sensitivity requirements can be withheld from library preparation. Pre-determining poor quality samples can save significant time and cost associated with library prep, sequencing and data analysis.
Additionally, information gathered from PreSeq can be used in a variety of optimization experiments. Various extraction methods can be benchmarked against each other at little cost to the lab. When used in tandem with Archer Analysis, library preparation and sample handling procedures can be optimized, and incidences of human and sequencing error can be identified. Information on the quality of the sample can also be a critical component of archival sample tracking.
The kit contains 24 reactions-worth of the following components:
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