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Article: Understanding Different MassARRAY® Chemistries

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Introduction

Molecular testing chemistriesThe MassARRAY® System is a multifaceted molecular testing platform with a wide array of applications. The system is compatible with testing for several types of genetic testing applications. The MassARRAY System is powered by several proprietary panels and chemistries, each designed to deliver targeted results for different diagnostic needs. These chemistries include iPLEX Pro, iPLEX HS, UltraSEEK, and Agena ClearSEEK.

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MassARRAY System Workflow & Flexible Chemistries

All chemistries on the MassARRAY System employ mass spectrometry and end-point PCR (Polymerase Chain Reaction) testing. After nucleic acid extraction, samples are processed through a simple workflow:

Molecular testing workflow

The MassARRAY System is powered by matrix-assisted laser desorption/ionization – time of flight (MALDI-TOF) for the detection of DNA molecules and distinguishes genetic variants. Each panel is designed to identify genetic variants according to their individual mass. The DNA or cDNA is amplified using one of several Agena Bioscience® chemistries, available through pre-designed or custom panels for specific customer needs.

iPLEX Pro Chemistry & Workflow

The robust multiplexed primer extension chemistry of the iPLEX assay combines with MALDI-TOF to analyze up to thousands of genotypes per day with a high level of accuracy and reproducibility. iPLEX single base extension chemistry enables highly stringent calling thresholds and superior performance at high multiplexing levels.1

iPLEX Chemistry

In the iPLEX process, genomic DNA or cDNA is amplified in 96- or 384-well microtiter plates using iPLEX reagents. Once PCR is complete, excess nucleotides are dephosphorylated by shrimp alkaline phosphatase (SAP). Next, the iPLEX single base extension reaction uses a mix of oligonucleotide extension primers to anneal the amplified DNA, which is added with an extension enzyme and terminators. Extension primers anneal directly adjacent to each SNP (Single Nucleotide Polymorphisms) site to be assayed and are extended and terminated by a single complementary base into the genotyping target site. Analytes are desalted and transferred from the microtiter plate via manual dispensation or an automated nanodispenser onto a SpectroCHIP® array, where they crystallize with a pre-spotted MALDI matrix.

After the process is complete, the SpectroCHIP Array is loaded into the MassARRAY analyzer, which detects DNA within a mass range of approximately 4,500 Da to 9,000 and distinguishes between analytes separated by 16 Da. Typer software generates a report identifying homozygous and heterozygous SNP alleles.

iPLEX Pro Sample Integrity Panels

Each of the iPLEX Pro Sample Integrity panels enable rapid and highly accurate sample identification and DNA quality measurements to generate a sample’s unique genetic fingerprint, which is used to deliver a Match or Mismatch result.

Sample ID panel comparison

The iPLEX Pro Sample ID panel is ideal for rapid and accurate sample management in molecular diagnostic labs, biobanks, and toxicology. This panel creates a unique genetic fingerprint for each subject, which is used to compare future samples to ensure the validity of the specimen or alert the laboratory that manipulation or mix-up has occurred.

The iPLEX Pro Exome QC panel also determines a sample’s identity, DNA quality, and DNA quantity. It targets 21 SNPs (Single Nucleotide Polymorphisms) in exonic regions of the genome and 3 biological gender markers. Additionally, it provides the number of intact, amplifiable DNA copies across a size range of 100, 200, 300, 400, and 500 nucleotides.

iPLEX HS Chemistry & Workflow

iPLEX HS panels on the MassARRAY System enable highly sensitive, multiplexed detection of genetic variants present in solid tumors. iPLEX HS detects clinically significant variants while minimizing sample rejection with single base extension enriched for mutant alleles in as little as one hour of hands-on time and a total of eight hours for results.

iPLEX HS uses as little as 10 nanograms of input DNA with high-sensitivity mutation detection for variants as low as 1% frequency. After PCR amplification of mutant variants, data output from the MassARRAY System distinguishes mutations from wild type signal, which is restrained to achieve the increased sensitivity. Clear spectra provide user-friendly, at-a-glance identification of actionable variants. The short amplicon length (80-120 bp) makes it ideal for use with degraded samples.

For solid tumors, iPLEX HS panels enable detection of variants as low as 1% MAF from FFPE tissue, core needle biopsies, FNA and cytology smears.

iPlex variant detection workflow

iPLEX HS Panels

iPLEX HS Panels are available for solid tumor profiling in several types of cancer, including:

The iPLEX HS Lung panel for tumor profiling of non-small cell lung cancer

  • Detect 70 clinically relevant variants across BRAF, EGFR, ERBB2, KRAS and PIK3CA
  • Identify variants at as low as 1% variant allele frequency
  • Use as little as 10 ng of DNA
  • 1 hour of hands-on time and 8 hours of total time

iPLEX HS Lung Panel Genes

The iPLEX HS Colon panel for tumor profiling of colorectal cancer

  • Detect more than 80 clinically relevant variants across BRAF, EGFR, KRAS, NRAS and PIK3CA
  • Identify variants at as low as 1% variant allele frequency
  • Use as little as 10 ng of DNA
  • 1 hour of hands-on time and 8 hours of total time

iPLEX HS Colon Panel Genes

The iPLEX HS Melanoma panel enables tumor profiling studies of melanoma specimens

  • Detect more than 90 clinically relevant variants from BRAF, KIT, NRAS, TERT and other genes
  • Identify variants as low as 1% variant allele frequency
  • Use as little as 10 ng of DNA
  • 1 hour of hands-on time and 8 hours of total time

iPLEX HS Melanoma Panel Genes

UltraSEEK Chemistry & Workflow

UltraSEEK® chemistry is ultrasensitive somatic mutation detection used for a variety of liquid biopsy needs. Highly sensitive with broad variant coverage. Multiplexed PCR followed by mutant specific single base extension and capture by streptavidin-labeled magnetic beads. Built-in controls verify the presence of DNA template in the reaction.

The MassARRAY System, powered by UltraSEEK chemistry, provides a targeted, multiplexed method for detecting rare events, increasing the sensitivity to less than 1% and as low as 0.1%. On the MassARRAY System, UltraSEEK identifies low-level genetic determinants for initiation and proliferation in primary tumors, early detection of metastasis, patient-specific mutation therapeutic monitoring, and orthogonal validation of next-gen sequencing or real-time PCR results to confirm or rule out borderline results.

UltraSEEK chemistry consists of PCRA amplification, like other panels on the MassARRAY System, of a set of pre-defined loci harboring the mutations of interest.

Liquid biopsy workflow

UltraSEEK Panels

UltraSEEK panels are available for solid tumor profiling in several types of cancer, including:

The UltraSEEK Lung panel enables study of disease progression and resistance in non-small cell lung cancer

  • Detect 70 clinically relevant variants across BRAF, EGFR, ERBB2, KRAS and PIK3CA
  • Identify variants at as low as 1% variant allele frequency
  • Use as little as 10 ng of DNA
  • 1 hour of hands-on time and 8 hours of total time

USK Lung Panel Genes

The UltraSEEK EGFR panel is used for residual disease and resistance monitoring studies in non-small cell lung cancer

  • Detect 6 clinically relevant EGFR variants including T790M, C797S, E746_A750del and L858R
  • Identify variants at as low as 0.1% variant allele frequency
  • Use as little as 10 ng of cfDNA
  • 1 hour of hands-on time and 8 hours of total time

USK EGFR Panel Genes

The UltraSEEK Colon panel is used for the study of disease progression and resistance in colorectal cancer

  • Detect over 100 clinically relevant variants across BRAF, EGFR, KRAS, NRAS and PIK3CA
  • Identify variants at as low as 0.1% variant allele frequency
  • Use as little as 10 ng of cfDNA
  • 1 hour of hands-on time and 8 hours of total time

USK Colon Panel Genes

The UltraSEEK Melanoma panel enables the study of disease progression and resistance in melanoma

  • Detect more than 55 clinically relevant variants across 13 genes including BRAF, KIT and NRAS
  • Identify variants at as low as 0.1% variant allele frequency
  • Use as little as 10 ng of cfDNA
  • 1 hour of hands-on time and 8 hours of total time

USK Melanoma Panel Genes

Agena ClearSEEK Chemistry & Workflow

Agena ClearSEEK™ chemistry is a wild-type (WT) terminator-depleted chemistry designed to only generate extension terminator signal in a mutant DNA specimen. This allows for quantification of a mutation down to a very low variant allele frequency (VAF) as the analytical window is not dominated by the wild-type allele. To confirm wild-type sample amplification an extension primer for each wild-type PCR amplicon is included in the panel. Following the PCR reaction, SAP addition, and Agena ClearSEEK extension reaction, the samples were desalted by ion exchange resin treatment, spotted onto SpectroCHIP Arrays and interpreted on Typer Software.

Agena ClearSEEK chemistry detects variants as low as 1% minor allele frequency and is compatible with multiple sample sources, including formalin-fixed, paraffin embedded tissue with less than 10% tumor burden.2

Agena ClearSEEK PIK3CA Panel

The Agena ClearSEEK PIK3CA Panel targets a set of 20 variants in PIK3CA associated with response to targeted therapy, including the activating mutations recommended by the National Comprehensive Cancer Network Guidelines.3

The Agena ClearSEEK PIK3CA panel enables tumor profiling studies of breast cancer

  • Detect 20 clinically relevant variants from PIK3CA
  • Identify variants at as low as 1% variant allele frequency
  • Use as little as 10 ng of DNA
  • 1 hour of hands-on time and 8 hours of total time

Agena ClearSEEK Breast Panel Genes

Custom Panels

In addition to the panels mentioned above, Agena Bioscience also offers custom panels unique to individual customer needs. The iPLEX Pro, iPLEX HS, UltraSEEK, and Agena ClearSEEK chemistries are available for use in these custom assays, which are developed in coordination with each laboratory. Learn more about our customers’ success stories.

References:
1. Gabriel S, Ziaugra L, Tabbaa D. (2009) SNP genotyping using the Sequenom MassARRAY iPLEX platform. Curr Protoc Hum Genet 60:2.12.1-2.12.18. 2. AH Box et. al. Evaluation of a Mass Spectrometry-Based PIK3CA Mutation Assay for Predictive Breast Cancer Therapeutic Decision Making. Poster session presented at: Association of Molecular Pathology Annual Meeting; 2020. 3. National Comprehensive Cancer Network (NCCN) Guidelines – Breast Cancer. Version 2.2022.