Overview

NuProbe’s Quantitative Amplicon Sequencing (QASeq) technology allows rapid (4-8 week) development of custom NGS panels to detect mutations at 0.2% Variant Allele Frequency (VAF) and deletions at 1.97 ploidy or amplifications at 2.04 ploidy from FFPE tissue, fresh/frozen tissue or blood cfDNA.

Deletions and amplifications of genes, also known as copy number variations (CNVs), are present in a significant percentage of tumors, between 3% and 98% depending on the cancer type, and are clinically relevant as prognostic markers and as therapeutic targets.

Key Features

  • Highly multiplexed amplicon molecular barcoding: QASeq utilizes unique molecular identifiers(UMI) and PCR to accurately quantify SNVs and Indels as well as to detect CNV.

  • High conversion yields and proprietary bioinformatics: QASeq technology has a high conversion yield (median >60%), allowing sensitive detection from small sample inputs (30ng FFPE DNA, 8ng fresh/frozen DNA, 10ng cfDNA). The fastq-to-variant report for related panels includes CNV and mutation calls with a user-friendly cloud or locally-run analysis program.

  • Simultaneous detection of mutations, deletions, and CNVs: QASeq technology allows the detection of mutations at 0.2% VAF and deletions at 1.97 ploidy (3% tumor cells with 1 copy deletion) or amplifications at 2.04 ploidy (4% tumor cells with 1 copy amplification) from FFPE tissue, fresh/frozen tissue or blood cfDNA.

  • Rapid development of custom NGS panels: QASeq technology allows rapid (4-8 week) development of custom NGS panels covering 1-20 genes.

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Mechanism

  • QASeq Mechanism

  • QASeq uses partially nested PCR to scales to high multiplexing to allow accurate CNV quantitation, while maintaining high on-target rate and high mutation sensitivity.

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Performance

  • Amplicon ploidy for a healthy PBMC donor (top) and for a breast cancer fresh/frozen (FF) tumor section (bottom).

  • Sample CNV call results for DNA from an ERBB2 CNV positive breast cancer patient (fresh/frozen tumor sample), and the DNA from the peripheral blood mononuclear cells (PBMC) from a healthy donor.

  • 8 ng of input DNA was used for each library. Genes with called CNVs are displayed in red. Genes within expected variation are shown in blue.

  • Comparison to IHC and ddPCR N = 18 patient FFPE tissue (breast cancer)

  • QAseq is more sensitive than ddPCR

  • QAseq detects deletions and mutations, IHC does not

  • QAseq analyzes thousands of loci, ddPCR analyzes 1 locus

  • Observed conversion yields for 179-plex QASeq panel, using 10 ng Horizon Multiplex I Wild Type cfDNA Reference Standard.
  • Because QASeq labels the top and bottom strands of a DNA molecule with different UMIs, the number of distinct UMI families roughly be double that of the observed double-stranded DNA molecules.
  • Conversion yield is calculated as observed molecule number (i.e. number of distinct UMI families divided by 2), divided by input molecule number.