Overview of techniques in Molecular Diagnostics ELKE BOONE 23 MAART 2017

Transcription

1 Overview of techniques in Molecular Diagnostics ELKE BOONE 23 MAART 2017

2 Molecular Diagnostics

3 Molecular Diagnostics?

4 Molecular Diagnostics

5 FISH or CISH techniques

6 FISH or CISH techniques

7 FISH or CISH techniques

8 FISH or CISH techniques

9 PCR techniques

10 Reverse transcriptionpcr techniques

11 RNA molecules

12 Cell-free DNA molecules Liquid biopsies come of age in molecular diagnostics

13 Cell-free DNA molecules Foetal, placentaldna canbefound in bloodof mother

14 Cell-free DNA molecules cfdnaor cffdnashouldbeseenas RNA! Half-life in circulation is between 16 min and 2,5 hours Cleared via nuclease action and renal extraction into the urine cfdnafromtumor cellsor placenta is smaller thancfdnaof the circulating blood cells Special blood collection tubes needed! 166bp

15 DNA or RNA isolation

16 DNA or RNA isolation

17 DNA or RNA isolation

18 DNA or RNA isolation

19 DNA or RNA isolation Chemistry determines which DNA size is isolated!

20 cdna synthesis

21 PCR techniques

22 PCR techniques

23 PCR techniques

24 PCR techniques

25 PCR techniques

26 PCR techniques Digital PCR More sensitive? Absolute quantification No need for standards

27 OLD school Sanger sequencing 1 PCR per sample andper target DNA region > 10 6 similar DNA fragments Accumulation of fluorescent signals originating from different DNA fragments with a same base length

28 OLD school Sanger sequencing Single nucleotide difference 10% SNP Heterozygote SNP (50%) 3% SNP

29 OLD school Sanger sequencing Mixed electropherogram(e.g. insertions/deletions) V A I K E L R E A T S GTC GCT ATC AAG GAA TTA AGA GAA GCA ACA TCT GTC GCT ATC AAA ACA TCT V A I K T S

30 OLD school Sanger sequencing Pitfalls Sanger sequencing Low throughput 1 gene region= 1 PCR = 2 sequencingreactions Labor intensive 50 target regions x 10 samples x 2 sequencing reactions = 1000 reactions) Low sensitivity Depending on sequencing context 10% to 25% Exact % of variant cannotbe determined Very difficult identification of deletions/insertions

31 NGS advantages.. as compared to Sanger sequencing Different molecules can be sequenced together in one sequencing run(1 cluster = 1 original DNA molecule = 1 read, millions of reads per run) Different patient samples can be pooled (index sequencing) High throughput sequencing: different outputs possible due to different sequencing cells (12 to 50 million reads, up to 15 gigabases) samples sequencing dept = coverage = number of reads at a specific base position Deep sequencing (MRD): very few samples and amplicons very high sequencing depts Higher sensitivity depending on read depth and DNA region: 5% with read depths of around 1000 molecules (50 variant reads/1000 total reads) 0,5% with read depths around molecules (50 variant reads/10000 total reads) Determination of the variant allele frequency (VAF)

32 Next Generation Sequencing Clonal Amplification

33 Next Generation Sequencing 1 Genomic DNA for sequencing 3 Picture 1 PCR Ampliconsof target regions Picture 2 PCR Amplicons with sequencing adaptors 2 1 bead= 1 sequence 1 cluster = 1 sequence High-throughput= many molecules sequenced together Picture 1 Clonal amplification to generate enough identical signalsper beador per cluster 4 Picture 2 Ion Torrent GS junior MiSeq(Illumina)

34 Index and paired-end NGS Generation of DNA amplicons Addition of patient indices and Sequencing primers

35 Library prep strategies R. Simon and S. Roychowdhury, Nature Reviews Drug Discovery, 2013

36 Bio-informatics A new technique in molecular diagnostics

37 Which technique, when?

38 The future goes smaller and quicker Extraction/PCR in one cartridge, put on a specific analyzer

39 The future goes smaller and quicker Nanomedicine: diagnosis, monitoring, therapy.

40 The future goes smaller and quicker Lab-on-a-chip (eg IMEC)

41 Toboldlygo.. DNA comparison scans? Whereno onehas gonebefore!