High throughput genotyping and mutation scanning

1. High throughput genotyping and mutation scanning Zoe Allen 09.11.09

2. Definitions • High throughput genotyping • Looking for a known mutation • Mutation scanning • Needle in haystack

3. HT Genotyping • For example – haemochromatosis mutations • C282Y • H63D • Many samples received in labs • Methods available for high throughput mutation detections • Real time PCR • Pyrosequencing • High resolution melt curve analysis • Not going to discuss RTPCR or pyrosequencing as most people probably know the basics

4. Pre-screening v full gene sequencing • Less samples to be sequenced in total • Cost • Pre-screening often cheaper than full gene sequencing • Analysis • More simplified analysis with prescreening • Less data to be checked • Processing steps • Sequencing – can be time consuming, many stages from DNA  end sequence • Pre-screening – methodology has less hands on stages

5. CSCE • Conformation sensitive capillary electrophoresis • Basically fluorescent heteroduplex analysis • BRCA1 and BRCA2 designed by NGRL Wessex • High throughput method on ABI analysers • Depending on size of capillary array • Need a modified polymer • More sensitive than DHPLC

6. CSCE method

7. Advantages Different fluorescent dyes enable multiplexing More sensitive than DHPLC Cheaper than sequencing Less complex analysis Tagged primers can be used for sequencing Easily automatable Disadvantages Loss of resolution when performing long runs – ABI needs a rest! Requires a modified conformational analysis polymer Home made ABI don’t like to support Machine used for CSCE cannot be used for sequencing Need to change array May need post PCR processing to dilute samples CSCE

8. HRM • Can be used for • Mutation scanning • E.g. BRCA1 and BRCA2 • Genotyping known mutations/SNPs • Methylation analysis • Detection of somatic mutations • Bruder et al (2008) used

9. HRM DNA is saturated with dye molecules DNA is then melted Temperature at which it melts depends on the fragment composition If a variant is present it will melt differently to WT fragment

10. HRM

11. Advantages Closed tube method No post PCR processing required Non destructive – can use same products for sequencing/gel electrophoresis Can design melt probes for frequent SNPs – reduce sequencing required Can detect mitochondrial variants at levels ranging from 1-100% heteroplasmy = sensitive Can determine percentage of methylation Can detect level of somatic mutations e.g. in colon cancer 384 samples in a single five to 15 minute run Disadvantages Fragment size is small 150-200bp optimum Cannot have too many melt domains or a loss in sensitivity is seen Well-well variations (can be overcome using temperature validation probes) Shape of melt curve is not good enough to reliably identify a variant – need to add specific melt probe Homozygous variant detection is poor Perform heteroduplex mixing experiments Not easily multiplexed HRM

12. Detection of somatic mutations - HRM • Used a dilution series as a ruler to determine the level of somatic mosaicism • Accurate to a level of 5-10% • Data matched that of pyrosequencing • Assay was less expensive and simpler to perform • If specific melt probes are included the detection improves to below 5%

13. Sequencing grade screening for BRCA1 variants by oligo-arrays • Designed a BRCA1 specific array covering entire coding region • 1423 oligo probes arranged at 4-nucleotides tiling based on arbitrarily selected BRCA1 sequence • Oligo probes varied in size (18-25nt) to maintain constant melting temperature • 38 additional probes for known BRCA1 variants added • Method • Probes spotted onto array slide – 3’ amine modification • DNA extracted and whole gene PCR’d in 11 fragments • T7 promoter attached to F primer to allow in vitro transcription • Labelled cDNA co-hybridised onto BRCA1 chip • Hybridized arrays scanned

14. Sequencing grade screening for BRCA1 variants by oligo-arrays • Results compared to sequencing • 85 patients tested • only confirmed oligo array’s accuracy by testing 1 fragment of BRCA1 • Paper suggests ‘whole BRCA1 gene can be analyzed with one oligo array reaction and have the same accuracy as at least 70 sequencing reactions (about 35kb)’ • Probably not a valid method for our laboratories at this time but a nice insight into alternatives to sequencing

15. Mass Spectrometry • Wang et al (2002) – analysis by MS of 100 CF gene mutations in 92 patients with CBAVD • MALDI-TOF mass spectrometry • Matrix assisted laser desorption ionization time of flight Normal alleles at all 3 loci Homozygous p.Phe508del Heterzygous p.Phe508del

16. Advantages High throughput specific genotyping Read length up to 800bp Cost effective No product purification needed Automated data analysis Analyze up to 3072 reactions/day (Sequenom) Disadvantages Initial cost of machine very high Sample must be very pure Process sounds like a lot of work but can’t really comment! Mass Spectrometry More suited to detection of known variants than mutation scanning

17. References • Human Mutation – May 2009 • Special edition all about HRM! • Jimenez et al (2009), Clinical Biochemistry, 42:1572-1576 • Advantages of the HRM in the detection of BRCA1 and BRCA2 mutation carriers • Vossen et al (2009), Human Mutation, 30:860-866 • High resolution melting analysis (HRMA) – more than just sequence variant screening • Wittwer (2009), Human Mutation, 30:857-859 • High resolution DNA melting analysis: Advancements and limitations • Monaco et al (2008), Journal of translational medicine, 6:64 • Sequencing grade screening for BRCA1 variants by oligo arrays • Wang et al (2002), Human Reproduction, 17:2066-2072 • Analysis by mass spectrometry of 100 CF gene mutations in 92 patients with CBAVD