Overview

Structural variants involving deletions and amplifications of large sections of DNA (>1 kb), known as copy number variations (CNVs), are present in a wide range of cancer types and are clinically relevant as prognostic markers and as therapeutic targets. While next-generation sequencing (NGS) methods can sensitively detect single nucleotide variants (SNVs) and small insertions or deletions (indels) below 1% variant allele frequency (VAF), limits of detection for CNVs are significantly worse- around 25% tumor load with a heterozygous single copy gain or loss. Due to this lack of sensitivity, methods such as immunohistochemistry (IHC) or in situ hybridization are generally preferred for CNV detection in clinical settings. However, these methods are low throughput and not applicable to liquid biopsy samples, including peripheral blood mononuclear cells (PBMCs) and cell-free DNA (cfDNA). Additionally, these other methods are not able to detect SNVs and indels at low VAF.

NuProbe’s Quantitative Amplicon Sequencing (QASeq) technology is an NGS-based method that sensitively and quantitatively detects CNVs down to 5% heterozygous single copy gain or loss. QASeq simultaneously detects SNVs and indels at 0.2% VAF from a range of sample types, including formalin-fixed paraffin-embedded (FFPE) tissue, fresh frozen (FF) tissue, PBMCs, and cfDNA. This sensitivity is enabled through a combination of unique molecular identifiers (UMIs) and a highly multiplexed, partially-nested PCR primer design which results in high conversion yield, with around 65% of the DNA molecules in a sample labeled with high on-target rates. As a result, less DNA is needed to make these calls.

Key Features

  • Sensitive and simultaneous detection of CNVs, SNVs, and indels: QASeq detects CNVs down to 5% heterozygous single copy gain or loss and SNVs and indels at 0.2% VAF
  • Quantitative: CNVs, SNVs, and indels are accurately quantified through the use of UMI labeling and NuProbe’s proprietary bioinformatics pipeline
  • Compatible with a range of specimen types, including liquid biopsy: Can be used with FFPE tissue, FF tissue, PBMCs, or cfDNA
  • Lower input DNA required: QASeq’s high conversion yields (around 65%) and on-target rates mean that less input DNA is required to make these calls (30ng FFPE, 8ng FF, 10ng cfDNA)

Mechanism

  • QASeq Mechanism

  • QASeq starts with a forward primer containing a UMI sequence. After the initial UMI addition step, universal primers are used to pre-amplify the desired amplicons. A partially-nested reverse primer is used in the next PCR step to improve the on-target rate by selecting against primer dimers and off-target genomic amplicons. The DNA library subsequently follows standard index PCR steps.

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.