TLA for FFPE Tumour Samples

The Targeted Locus Amplification (TLA) technology uses crosslinking and the physical proximity of nucleotides within a locus of interest as the basis of selection. As such, TLA presents particular advantages in crosslinked FFPE samples.

In the TLA technology for FFPE tumour samples, crosslinked DNA fragments are religated to generate TLA template: combinations of religated DNA fragments that prior to formaldehyde crosslinking occurred in extreme physical proximity to each other and therefore originated from the same genomic locus. TLA template is used as input material for enrichment and targeted sequencing. In this manner, TLA in combination with conventional capture or linear PCR-based enrichment, enables the enrichment of all sequences that originated from the same locus.

As a result, the TLA FFPE protocol uniquely enables the targeted complete sequencing and the detection of all single nucleotide and structural variants in genes of interest.

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  TLA 1

  DNA in FFPE material is crosslinked and fragmented.

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  TLA 2

  Crosslinking very preferentially occurs between sequences that occur in extreme physical proximity and therefore between sequences originating from the same genomic locus.

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  TLA 3

  Sequences from a genomic region of interest (depicted in red) will thus be very preferentially crosslinked to each other in FFPE samples.   

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  TLA 4

  In the TLA protocol, crosslinked DNA fragments are ligated to form TLA template; stretches of DNA consisting of multiple ligated DNA fragments originating from the same genomic region. 

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  TLA 5

  As a result of stochastic differences in DNA folding and in the DNA crosslinking, fragmentation and ligation steps, TLA template consists of a repertoire of ligated DNA sequences composed of different combinations of DNA fragments.

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  TLA 6

  Using locus specific primers/probes, TLA template fragments from a genomic region of interest are enriched with either (multiplex) linear PCR and/or multiplex capture.

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  TLA 7

  As a result, the complete genomic region is enriched and can be sequenced using next generation sequencing technologies. 

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  TLA 8

  In this manner the TLA technology enables targeted hypothesis-neutral sequencing. It detects all sequence and structural variants in loci of interest.

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  TLA 9

  In combination with multiplex PCR and/or capture any combination of regions of interest can be enriched and sequenced.