Software programs for sequence variant detection and interpretation

1. Software programs forsequence variant detection and interpretation Huong Le Department of Molecular & Clinical Genetics, Royal Prince Alfred Hospital

2. Sequence variant detection SOFTWARES Sequence variant interpretation SOFTWARE

3. Sequence variant detection softwares • AB software • Seqscape software using the KB Basecaller algorithm (v1.2) integrated with the Phred software provide quality value calculation per base. • Seqscape software contains many features such as • base calling program • Base calls • Measure quality value (QV) • Identify pure and mixed bases • Trim quality sequence • Filter out the poor sequence • sequence assembly, alignment and comparison • Search library for allele match • Generate reports • SoftGenetic software is a pioneer in mutation detection from DNA sequence traces • Mutation surveyor software use direct sequence trace comparison method (the anti-correlation method) to locate • Heterozygote / Homozygote point and indels mutations • The software remove the effect of base calling error and reduced the fault positive rate • as the rate of human mutation is ~1/1200 or 0.08% ) and basecalling accuracy as Phred score of 18-20 is approximately to 98-99% • Therefore sensitive for detection of rare variants that present as low as 10% of the population using the 2Dir smaller peaks • Robust sequence aligment Seqscape v2.5 Mutation surveyor

4. Seqscape and cDNA annotation set up

5. Seqscape and cDNA annotation set up

6. Seqscape – Genotype Table c. 681 location g. 2479065 location

7. Mutation Surveyor and the algorithm • Trace Comparison The DNA nucleotide peak intensities of traces are normalised and aligned and then the actual traces are compared using the mathematical algorithm called anti-correlation Reference trace R Sample trace S Difference between the R and S trace green line

8. The anti-correlation algorithm • The anti-correlation method • The differences between reference and sample traces were calculated and note any found in the mutation electropherogram. At any specific location that reference and sample have the same type of bases and the anti-correlation will be zero otherwise this value will be very high if these bases are different at the same point

9. Mutation score • The four numbers on the mutation peak is the score, mutation peak height, overlapping factor and intensity dropping factor. • mutation peak height (the highest peak in mutation electropherogram) • Overlapping factor indicate the relative shift of the two peaks (reference/sample) of different colors in the horizontal direction • Intensity dropping factor indicate the vertical peak intensity has dropped relative to the neighboring peaks • Mutation score= -10log(erfc[(s/n)/√2])*dropping factor*overlapping factor • erfc: complementery error function

10. Mutation Surveyor and result output

11. Homozygous indel detection and robust alignment based on the three following methods • Rough alignment with sequence text: use sequence text to find the best match with reference sequence • Robust alignment with DNA migration time: when two sequences are matched, the DNA migration times slope of the two match sequence are generated. • Maximum correlation for local adjustment if insertion occur- a compression area will form and if a deletion occur the a gap will be formed so that bases from previous and after will still align with the reference sequence in order to illustrate the indels then the center line will turn red from green and a heavy line will draw above the point • Heterozygous indel detection • Using a method to de-convolute the heterozygous trace into two clean sets of sequence traces according to the reference trace • Use robust alignment to align good traces -> the same slope is generated for the overlapping region of mixture (shown roughly align to reference trace)

12. Mutation Surveyor and indel mutation detection sample trace Conserve trace identical to reference trace mutation trace left after reference subtraction Shift trace Deletion detection

13. Mutation Surveyor and heterozygous deletion case

14. Heterozygous deletion case

15. Mutation Surveyor and sensitivity • very sensitive for detection of rare variants which present as low as 10% of • the population using the 2Dir smaller peaks

16. Mutation Surveyor and analysis of multiple genes

17. Mutation Surveyor and TTR mutation detection

18. ALAMUT : Variant interpretation software • Developed by Interactive Biosoftware (IBS)-France

19. ALAMUT : software and database

20. ALAMUT : software and database

21. ALAMUT: information for navigation

22. How to select your gene of interest

23. LDLR GENE

24. Different sources of database can be linked

25. SNPs viewing

26. Different levels of search

27. Protein multi-alignment

28. Variant prediction tools

32. Result interpretation

33. Database links

34. Database links

36. ALAMUT : Add new mutation to database

37. Depienne, C., et al., Spectrum of SCN1A gene mutations associated with Dravet syndrome: analysis of 333 patients. J Med Genet, 2009. 46(3): p. 183-91. • Analysis SCN1A mutations in a large number of samples using • Seqcape v2.5 for sequence variant detection • Alamut v.131 software for • assessing mutation interpretation and amino acid conservation in orthologues • Use GD to test the effect of residue replacement with respect to a range of physicochemical properties (volume, charge and composition of side chain)

38. ALAMUT: software validation • The software was assessed in four areas • Its user interface and usability • The suitability of its data sources • Its applicability to diagnostic testing • Its validity and accuracy • The software was assessed by • National Genetic Reference Lab Manchester • Leiden University Medical Centre • GUY’S Hospital London • PRAGUE

39. References • EuroGentest Unit 5 on decision support for molecular diagnostic laboratories using Interactive Biosoftware Alamut prepared by NGRL Manchester • http://leedsdna.info/HUGO/2004/Lab_Notes/2004_labs.htm