Download Genotyping Errors

François Ancien
Sascha Kretzschmann
Olivier Suplis
Genotyping Errors
Causes, Consequences and Solutions
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Reference paper
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.
2.
3.
4.
5.
Introduction
DNA-related errors
Biochemical errors
Human errors
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) :
[1]
Often associated with restriction (endonuclease – "molecular scissors")
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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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How can errors appear during PCR ? Is it possible to reduce the
risk of errors ?
BIOCHEMICAL ERRORS
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Biochemical errors
• Often appear during PCR
• Often happen because of :
– Badly chosen polymerase
– Badly designed primers
[3]
• Consequences are :
– Null or false alleles
– Dependent of the study
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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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Which role play humans concerning genotyping errors? Do they
produce many errors? Are there possible solutions to avoid them?
HUMAN ERRORS
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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)
[8]
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Human errors (solutions)
• use automated scoring software (from Applied BioSystems)
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Human errors (solutions)
• use automated scoring software (from Applied BioSystems)
[9]
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Human errors (solutions)
• use automated scoring software (from Applied BioSystems)
[10]
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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)
[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 but
never trust it 100%
• error rates should be estimated and reported
in every study
[13]
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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-ideedessus-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 DNAbased population inventories. Mol Ecol. 2003;12:1375-87.
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