Cergentis’ Targeted Locus Amplification (TLA) technology constitutes a paradigm shift by, uniquely, enabling targeted and complete gene sequencing.
The TLA technology uses the physical proximity of nucleotides within a locus of interest as the basis of selection. First, DNA is crosslinked, fragmented and religated. A primer pair complementary to a small locus-specific sequence is then used to amplify tens of thousands of surrounding base pairs. In this manner, the complete sequence of a locus is amplified. Amplified loci are subsequently sequenced with Next Generation Sequencing technologies.
The TLA Technology enables targeted complete sequencing of any locus or (trans)gene of interest and allows for detection of all Single Nucleotide Variants (SNV's) and structural variants.
In combination with paired-end sequencing or long-read sequencing technologies, TLA also enables the comprehensive haplotyping of analysed loci.
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First, genomic DNA is crosslinked.
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Crosslinking preferentially occurs between sequences that occur in extreme physical proximity.
Crosslinking, therefore, results in the crosslinking of sequences from the same locus (depicted in red).
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The crosslinked DNA is fragmented, religated with a ligase enzyme and then decrosslinked.
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This results in TLA template; long stretches of DNA consisting of religated DNA fragments originating from the same locus.
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This template is fragmented and circularised.
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Stochastic variation in the folding, crosslinking and religation of DNA fragments in individual copies of a locus, results in a repertoire of DNA circles that are composed of unique combinations of DNA fragments from that locus.
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Circular fragments originating from the locus of interest are amplified with inverse primers complementary to a short locus-specific sequence.
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As a result, the complete locus is amplified and can be sequenced using Next Generation Sequencing technologies.
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In this manner, the TLA technology enables targeted hypothesis-neutral sequencing. It detects all sequence and structural variants in loci of interest, also in heterogeneous samples such as tumours.