The Archer® VariantPlex® CFTR kit is a targeted next-generation sequencing (NGS) assay to detect known and unknown variants in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Independent, unidirectional primers amplify large genomic regions, enabling detection of select intronic variants and exonic mutations as well as large deletions that would otherwise be difficult to detect with opposing primer techniques. Bidirectional coverage reduces allele dropout, increasing your chances of capturing previously unknown variants. This fast and easy-to-use lyophilized workflow is practical and economical for resource-limited communities and is thus suitable for global, pan-ethnic CFTR mutation profiling.
For Research Use Only. Not for use in diagnostic procedures.
Annotated mutations covered
# of GSP2s
Total target size
Input DNA required‡
Recommended # of reads†
Coverage uniformity > 20% mean†
blood, saliva, buccal cells,
†Expected coverage uniformity at the recommended read depth.
‡input recommendations for FFPE samples vary depending on Archer Preseq® DNA QC score. 50ng input recommended in absence of PreSeq screening
Cystic Fibrosis (CF) is an autosomal recessive disease that is characterized by the build up of thick mucus resulting in chronic lung infections and airway inflammation. Loss-of-function mutations in the CF transmembrane conductance regulator (CFTR) gene underlie CF, as CFTR has essential roles in chloride ion transport and the production of thin mucus. While there is no cure for CF, early diagnosis and treatment interventions are necessary to help prevent airway obstruction and lung infections (1). Therefore, carrier identification and newborn screening have significant implications in the overall prognosis of CF patients.
CF is currently under-diagnosed in nonwhite populations compared to white populations (3). This ethnic disparity in CF diagnosis is primarily attributed to differences in underlying CFTR mutations, which were recently shown to vary significantly across ethnic groups by Iris Schrijver et al. (4). For example, the Phe508del variant is present in 90% of whites, 83% of Native Americans, 70% of Hispanics, 59% of Asians and 62% of African Americans among those diagnosed with CF (3). Current CFTR genotyping assays were designed based on the ACMG and ACOG-recommended 23-mutation panel, which was generated from mutation profiles obtained from white individuals in a study that pre-dates the identification of mutation differences across ethnic groups (1,2). In nonwhite populations, other novel point mutations, duplications and deletions in CFTR are more prevalent (3). Due to these differences in mutation profiles between ethnic populations, an unbiased approach to detect both known and unknown variants is key for pan-ethnic CF diagnosis. Archer's Anchored Multiplex PCR (AMP™) technology provides superior coverage through the use of molecular barcoded adaptors (MBCs) and independent gene-specific primers (GSPs), enabling unbiased NGS-based detection of known and unknown mutations across larger regions of the CFTR gene.
The majority of mutations in the CFTR gene are base substitutions and deletions of varying sizes (1,5). The CFTRdele2,3(21kb) mutation, reported by Dörk et al. in 2000 results in loss of exons 2 and 3 in CFTR transcripts, producing a premature termination signal within exon 4. This severe deletion is more prevalent in Central and Eastern Europeans and confers an aggressive phenotype. As shown in the figure below, the anchored primers used in AMP allow for amplification into large genomic regions from both ends independently, allowing Archer libraries to sequence both wildtype and deletion alleles using the same primers. We tested the ability of the VariantPlex CFTR kit and Archer Analysis DNA anomaly pipeline to detect CFTRdele2,3(21kb) in a pre-validated DNA sample obtained from the NIGMS Human Genetic Cell Repository at the Coriell Institute for Medical Research: NA18668. Part (B) in the adjacent figure show the CFTRdele2,3(21kb) detected from this sample in the Archer Analysis DNA anomaly pipeline and sequence identification of the genomic breakpoint.
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 the risk of false negative mutation detection due to ethnic genetic polymorphisms.
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