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), or cell-free DNA (cfDNA). Additionally, these other methods and 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, meaning that less DNA is needed to make these calls.
- 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)