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

Genotyping Errors 1

François AncienSascha KretzschmannOlivier Suplis

Genotyping Errors

Causes, Consequences and Solutions

10.12.2013

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Reference paper

10.12.2013 Genotyping Errors

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

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Contents

1. Introduction

2. DNA-related errors

3. Biochemical errors

4. Human errors

5. Conclusion


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Introduction


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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)


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Introduction

The types of applications that involve genotyping are :– Population studies (assessment of population structure, size,

diversity, …)– Linkage analysis– Association studies– Individual identification (forensics, …)– …


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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


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Introduction

The experimental protocols based on these markers involve PCR amplification (polymerase) :


Often associated with restriction (endonuclease – "molecular scissors")

[1]

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Introduction


[2]

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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.


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DNA-related errors


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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


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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


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BIOCHEMICAL ERRORS

How can errors appear during PCR ? Is it possible to reduce the risk of errors ?


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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


[3]

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Biochemical errors

• How avoid them ?– Choose an adapted polymerase– Choose the primers wisely– Repetitions on different samples


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Biochemical errors

• How avoid them ?– Choose an adapted polymerase

• Some do less mistakes• Some are more resistant to temperature changes• Some are more processive


[4]

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Biochemical errors

• How avoid them ?– Choose the primers wisely

• Can’t do secondary structures• No complementarity between primers• GC percentage = +/- 50%


[5]

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Biochemical errors

• How avoid them ?– Repetitions on different samples

• Repetitions show errors that still appears

• Results statistically more valuable


[6]

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HUMAN ERRORS

Which role play humans concerning genotyping errors? Do they produce many errors? Are there possible solutions to avoid them?


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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– …


[7]

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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)


How exactly ?[8]

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Human errors (solutions)

• use automated scoring software (from Applied BioSystems)


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[9]

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[10]

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• 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 shouldbe kept in separate facilities– > strict rules governing movement of people

or material between them (facilities)


[11]

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Human errors (solutions, cont.)

• avoided completely typographical error in [Paetkau2003]– > with the help of databases– > built laboratory database around field database

direct information delivery automation


[12]

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CONCLUSION


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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 butnever trust it 100%

• error rates should be estimated and reportedin every study


[13]


Thank you for your attention! =)

Questions ?

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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


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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.


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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.


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

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