Methodologies
CTGT uses complementary methodologies to attain the most sensitive mutation detection results available:
1. DNA Sequencing
Connective Tissue Gene Tests’ many years of experience with DNA sequencing combined with its extensive mutation and polymorphism databases ensure very high test sensitivity.
A. Sanger Sequencing
Direct DNA sequencing of PCR products generated from genomic DNA is performed for all tests offered by Connective Tissue Gene Tests. In all instances, sequencing of exons and exon-intron boundaries is performed for all genes. This is the gold standard for mutation detection in genes and is highly sensitive for point mutations, splice site mutations, and small exonic deletions, insertions and indels.
B. Next Generation Sequencing
In addition to Sanger sequencing, Connective Tissue Gene Tests also offers Next Generation Sequencing (NGS) using the Illumina MiSeq platform. NGS is useful for disorders that can be caused by mutations in multiple genes. CTGT has a unique approach to NGS panels. Customizable panels are offered so that the submitting physician can order analysis of the genes most relevant to the patient's differential diagnosis. This approach provides the submitting physician considerable advantage over the large, unfocused and invariant panels offered by competing laboratories. The customized CTGT NGS panels require the selection of a minimum number of genes or alternatively, may be selected as preconfigured panels.
NGS covers exons and exon-intron boundaries. Similar to Sanger sequencing, this method will detect point mutations, splice site mutations, and small exonic deletions, insertions and indels. All new sequence variants identified by NGS will be confirmed by Sanger sequencing.
2. Deletion / Duplication Analysis
While extremely sensitive, DNA sequencing does not detect all types of mutations that may be clinically relevant, including some duplications and deletions. Following extensive evaluation of the different methodologies, Connective Tissue Gene Tests developed the CTGT High-Density Targeted (HDT) Array for CNV detection:
The CTGT High-Density Targeted (HDT) Array is a custom designed Agilent G3 SurePrint microarray used in conjunction with the Agilent microarray platform. The aCGH technology on which the design is based uses 60mer oligonucleotide sequences designed to selectively complement target areas with an extremely high degree of specificity. High resolution and sensitivity are achieved by assigning a number of probes to each exonic region to allow for a minimum of 3 probes within each 300-500 base section of the genomic DNA sequence, and frequently many more. Non-coding intervening sequences are targeted at a minimum with approximately half of the density used for exonic regions. Many intervening sequences are more densely covered. Each probe used in the design has been thoroughly validated by an in silico bioinformatics process and by in vitro testing and has proven to yield accurate and reproducible results. Though individual copy number values derived from single probes may be highly sensitive, a much higher level of sensitivity is attained by the corroborating data of many closely-spaced probes. The minimum CNV size detected by this high-density array is 300-500 nucleotides, which is the technical limit of the assay using these stringent parameters.
Connective Tissue Gene Tests remains actively involved in evaluating alternative methodologies for mutation detection. These methodologies will be considered for introduction as they mature and are proven reliable, accurate and superior or complementary to existing technology.