Connective Tissue Gene Tests uses complementary methodologies to attain the most sensitive mutation detection rates possible.
1. DNA SEQUENCING
CTGT performs DNA sequencing tests for hundreds of disorders. Years of experience with DNA sequencing for molecular diagnostics ensures very high test sensitivity. All coding exons and exon boundaries of the targeted genes are covered. Coverage typically extends from 40 nucleotides prior to the start of an exon (-40) to 20 nucleotides past the end of an exon (+20). Minimum coverage is -3 to +6.
A. Sanger Sequencing
Targets are amplified by PCR and sequenced using ABI3730 sequencers. Proprietary custom designed software is used to identify sequence variants. All potentially pathogenic variants are confirmed by additional Sanger sequencing. Sanger sequencing is considered the gold standard for mutation detection and is highly sensitive for the detection of point mutations, splice site mutations, and small insertions and deletions.
B. Next Generation Sequencing
CTGT also offers Next Generation Sequencing (NGS) panels using the SureSelect and Illumina MiSeq platforms. NGS provides high throughput sequencing of many genes simultaneously and is useful for disorders associated with multiple genes.
CTGT utilizes advanced technology and innovative, custom-designed software to perform next generation sequencing tests that surpass all expectations. Our next generation sequencing panels provide Sanger quality data with a rapid turn-around time at reduced cost. CTGT offers 100% coverage of targeted exons and exon boundaries, unsurpassed sensitivity for the detection of insertions and deletions, and complete selectivity in the presence of pseudogenes.
All coding exons and exon boundaries of the genes on the panel are captured using SureSelect target enrichment. The products are sequenced on an Illumina MiSeq and proprietary, custom designed software is used to identify sequence variants. Exons that are not completely covered by at least 40 NGS reads are sequenced using Sanger sequencing. Additionally, all potentially pathogenic sequence variants identified by NGS are confirmed by Sanger sequencing. Similar to Sanger sequencing, this method will detect point mutations, splice site mutations, and small insertions and deletions.
2. DELETION / DUPLICATION ANALYSIS
CTGT utilizes two methodologies to detect deletions and duplications: a custom High-Density Targeted (HDT) Array and NGS based methodology for copy number variation (CNV) analysis. Both methodologies have a high resolution, and an extremely high degree of specificity and sensitivity. CNV detection limit is typically a single exon by both methodologies.
CTGT remains actively involved in evaluating new methodologies for mutation detection. These methodologies will be considered for introduction if they are proven to be reliable, accurate and superior or complementary to existing technology.