1. François Ancien Sascha Kretzschmann Olivier Suplis Genotyping Errors Causes, Consequences and Solutions 10.12.2013 1 Genotyping Errors

2. Reference paper 10.12.2013 Genotyping Errors 2 Pompanon F, Bonin A, Bellemain E, Taberlet P. Genotyping Errors: Causes, Consequences and Solutions, Nat Rev Genet. 2005 Nov;6(11):847-59.)

3. Contents 1.Introduction 2.DNA-related errors 3.Biochemical errors 4.Human errors 5.Conclusion 10.12.2013 Genotyping Errors 3

4. Introduction 10.12.2013 Genotyping Errors 4

5. Introduction Genotyping is the process of determining the alleles inherited by an individual at one or more loci. An allele is one of several alternative forms of the DNA sequence at a specific chromosomal location (i.e. locus) 10.12.2013 Genotyping Errors 5

6. Introduction The types of applications that involve genotyping are : –Population studies (assessment of population structure, size, diversity, …) –Linkage analysis –Association studies –Individual identification (forensics, …) –… 10.12.2013 Genotyping Errors 6

7. Introduction Information about allelic variation is obtained using molecular markers, that will show some degree of polymorphism among individuals. The article focuses on three types of markers : –Microsatellites : short sequences of 2-10 base pairs, repeated 3 to 100 times (alleles differ by the number of repetitions = length polymorphism) –AFLPs (amplified fragment length polymorphisms) : restriction fragments, ligated to adaptors and selectively amplified (presence/absence polymorphism) –SNPs : single nucleotide polymorphisms at known positions in the genome 10.12.2013 Genotyping Errors 7

8. Introduction The experimental protocols based on these markers involve PCR amplification (polymerase) : 10.12.2013 Genotyping Errors 8 Often associated with restriction (endonuclease – "molecular scissors") [1]

9. Introduction 10.12.2013 Genotyping Errors 9 [2]

10. Introduction One might think that ''DNA never lies'', but any experimental protocol is subject to errors. These are often overlooked in studies involving genotyping. The lack of standardized metrics makes it difficult to assess the quality of the results, or to make comparisons between studies. 10.12.2013 Genotyping Errors 10

11. DNA-related errors 10.12.2013 Genotyping Errors 11

12. DNA-related errors A mutation in the primer target sequence may prevent amplification. This will cause null alleles (i.e. not observed). An insertion or deletion close to a microsatellite marker can create size homoplasy (alleles that are the same size, thus scored as a single one). > Solutions : use of other markers, or rejection of samples 10.12.2013 Genotyping Errors 12

13. DNA-related errors Insufficient quality or quantity of DNA : [ for example in non invasive studies, where samples are collected from hair, … ]  Mistaken alleles : if contaminant molecules are amplified at the same rate as the target DNA molecules.  Allelic dropout : if too few target DNA molecules are present in an extract, due to dilution or degradation, or if the presence of inhibitors prevents restriction or amplification. > Solutions : multi-tube approach, or targeted re-analysis 10.12.2013 Genotyping Errors 13

14. BIOCHEMICAL ERRORS How can errors appear during PCR ? Is it possible to reduce the risk of errors ? 10.12.2013 Genotyping Errors 14

15. Biochemical errors Often appear during PCR Often happen because of : –Badly chosen polymerase –Badly designed primers Consequences are : –Null or false alleles –Dependent of the study 10.12.2013 Genotyping Errors 15 [3]

16. Biochemical errors How avoid them ? –Choose an adapted polymerase –Choose the primers wisely –Repetitions on different samples 10.12.2013 Genotyping Errors 16

17. Biochemical errors How avoid them ? –Choose an adapted polymerase Some do less mistakes Some are more resistant to temperature changes Some are more processive 10.12.2013 Genotyping Errors 17 [4]

18. Biochemical errors How avoid them ? –Choose the primers wisely Can’t do secondary structures No complementarity between primers GC percentage = +/- 50% 10.12.2013 Genotyping Errors 18 [5]

19. Biochemical errors How avoid them ? –Repetitions on different samples Repetitions show errors that still appears Results statistically more valuable 10.12.2013 Genotyping Errors 19 [6]

20. HUMAN ERRORS Which role play humans concerning genotyping errors? Do they produce many errors? Are there possible solutions to avoid them? 10.12.2013 Genotyping Errors 20

21. Human errors (error types) according a specific study [Hoffman2005]: 90% human factors –mixed up samples –contamination –inappropriate protocols –calling / scoring errors –data handling / processing errors –… 10.12.2013 Genotyping Errors 21 [7]

22. Human errors (proposals) avoid making errors involve only well-trained scientists or technicans use only standardized and validated procedures more (semi-)automations (e.g. pipetting, allele scoring) 10.12.2013 Genotyping Errors 22 [8]

23. Human errors (solutions) use automated scoring software (from Applied BioSystems) 10.12.2013 Genotyping Errors 23

24. Human errors (solutions) use automated scoring software (from Applied BioSystems) 10.12.2013 Genotyping Errors 24 [9]

25. Human errors (solutions) use automated scoring software (from Applied BioSystems) 10.12.2013 Genotyping Errors 25 [10]

26. Human errors (solutions) use automated scoring software (from Applied BioSystems) don’t leave critical human intervention/tasks to a single person –> at least two how at least one is highly experienced –> in [Paetkau2003] scoring results verification DNA samples and amplified DNA were should be kept in separate facilities –> strict rules governing movement of people or material between them (facilities) 10.12.2013 Genotyping Errors 26 [11]

27. Human errors (solutions, cont.) avoided completely typographical error in [Paetkau2003] –> with the help of databases –> built laboratory database around field database direct information deliveryautomation 10.12.2013 Genotyping Errors 27 [12]

28. CONCLUSION 10.12.2013 Genotyping Errors 28

29. Conclusion best way to reduce errors in genotyping experiments is to target human errors first working with protocols is essential combining multiple error detection techniques is necessary try to automate as much as possible but never trust it 100% error rates should be estimated and reported in every study 10.12.2013 Genotyping Errors 29 [13]

30. 10.12.2013 Genotyping Errors 30 Thank you for your attention! =) Questions ?

31. References Images [1] The Biotechnology Revolution: PCR and the Use of Reverse Transcriptase to Clone Expressed Genes. Leslie A. Pray, Ph.D. © 2008 Nature Education [2] Weber J L, May P E. Abundant class of human DNA polymorphisms which can be typed using the polymerase chain reaction. Am J Hum Genet. 1989;44:388-96. [3] http://commons.wikimedia.org/wiki/File:Pcr_machine.jpg [4] http://en.wikipedia.org/wiki/File:PDB_1xhz_EBI.jpg [5] http://commons.wikimedia.org/wiki/File:RNA_conservative_replacement.svg [6] http://www.clipartsfree.net/clipart/1599-cycle-icon-clipart.html [7] http://commons.wikimedia.org/wiki/File:Red_triangle_alert_icon.png [8] http://us.123rf.com/450wm/amasterpics123/amasterpics1231301/ amasterpics123130100010/17437646-3d-homme-pensant-avec-ampoule-idee- dessus-de-sa-tete-sur-fond-blanc.jpg 10.12.2013 Genotyping Errors 31

32. References [9] https://products.appliedbiosystems.com/ab/en/US/adirect/ab [10] https://products.appliedbiosystems.com/ab/en/US/adirect/ab;jsessionid= LL1LSXcPfHz2q2687dbL1ZZM7LWJfKBZNj313np8f5NjmvqWtp1n!693817876? cmd=catNavigate2&catID=600743 [11] http://farm3.staticflickr.com/2258/1681874529_563bd32140_o.jpg [12] http://fc00.deviantart.net/fs71/i/2013/016/f/7/layered_database_source_documents_ by_barrymieny-d5rnycs.jpg [13] http://www.ecrireundiscours.com/wp-content/uploads/2012/07/conclure-un discours.gif. 10.12.2013 Genotyping Errors 32

33. References Literature [Hoffman2005] Hoffman J I, Amos W. Microsatellite genotyping errors: detection approaches, common sources and consequences for paternal exclusion. Mol Ecol. 2005;14:599-612. [Paetkau2003] Paetkau D. An empirical exploration of data quality in DNA- based population inventories. Mol Ecol. 2003;12:1375-87. 10.12.2013 Genotyping Errors 33