The Archer® VariantPlex™ Assay system generates target-enriched libraries for next-generation sequencing (NGS) from DNA inputs. VariantPlex assays enable accurate and comprehensive detection of copy number variations (CNVs), single-nucleotide variants (SNVs) and insertions and deletions (indels) from low-input FFPE and other clinical sample types.
All VariantPlex kits include gene-targeting primers, easy-to-handle lyophilized enzymes, Illumina® platform-specific reagents to generate targeted sequencing libraries and an Archer PreSeq DNA QC Assay to confirm your input material quality prior to library generation.
Archer MBCs are also required, but provided separate for customizable multiplexing and indexing flexibility.
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Archer VariantPlex Assays are powered by powerful Anchored MultiPlex PCR (AMP) technology, which is uniquely suited for variant detection from FFPE samples.
Ligating half-functional adaptors to input DNA fragment ends enables single-direction priming and amplification into the adapter. Unidirectional priming is superior over opposing primer techniques because of the following:
Unique start sites
Because each fragment has a unique start site opposing the gene-specific primer, the molecular complexity of the sample is maintained through amplification and sequencing. With amplicon-based techniques, small, degraded fragments common in FFPE samples can fail to extend to both primers and are lost. Fragments that are of sufficient length to amplify become indistinguishable from one another. Resultant data fails to represent the sample complexity and has less accuracy in detecting low allelic frequency variants. Anchored Multiplex PCR, however, enriches fragments of all sizes, resulting in greater target recovery and complex libraries containing unique barcoded fragments.
AMP enrichment is unrestrained by opposing primers, allowing larger genomic regions to be amplified and sequenced. AMP-enabled long-range enrichment can cover more relevant variants without using multiple tubes and the additional cost, time and sample requirements associated with it.
Primer design flexibility
Longer regions of enrichment and strand specificity provide the flexibility to design primers that are optimized for coverage across challenging regions of the genome (e.g., GC-rich regions). Designs also include intronic buffers, so primers do not cover any portions of the target exon.
Dual independent coverage
Allele dropout due to primers blocking SNVs can reduce confidence in mutation calling. AMP’s strand-specific priming, however, facilitates dual independent coverage across target regions to ensure that some reads are retained when one primer drops out, thus reducing inconclusive results.
AMP attaches a unique tag to each input molecule prior to amplification. After sequencing, PCR duplicates, artifacts and sequencing errors can be identified and corrected. De-duplication and identification of unique reads enables robust, quantitative data that can be used for accurate variant calling and sensitive, concurrent CNV detection.
Sensitive and specific variant calling
Deduplication and error correction that accounts for PCR bias deliver a more accurate measure of allelic frequency and thus more accurate variant calls. In addition, false-negative variant calls due to low allelic frequency are reduced, because sample-specific sensitivity can be accurately measured by powerful MBC-enabled metrics. When used in conjunction with the PreSeq DNA QC Assay, sample-specific sensitivity can be predicted prior to library preparation and verified in Archer Analysis post-sequencing.
Robust CNV calling
By counting molecular barcodes within target regions and normalizing across the other targets or to a tumor normal sample, Archer Analysis reports relevant copy number variations present in the sample. CNV calls from Archer Analysis are sensitive, picking up fold changes below 2x that are highly correlated to qPCR and aCGH results. Learn more about AMP-enabled CNV detection in clinical sample types.
The VariantPlex kit provides all the reagents and accessories necessary for targeted library preparation. The kit is completely lyophilized, includes color coded tubes and only requires 2.5 hours hands-on time and 6 hours total preparation time. The tech-friendly protocol is a simple series of liquid transfers and eases reproducibility and contamination concerns.
Archer Analysis is a complementary software suite that provides in-depth sequencing data analysis and interpretation. Features of Archer Analysis include:
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