Genotyping errors occur when the genotype determined after molecular analysis does not correspond to the real genotype of the individual under consideration. Virtually every genetic data set includes some erroneous genotypes, but genotyping errors remain a taboo subject in population genetics, even though they might greatly bias the final conclusions, especially for studies based on individual identification. Here, we consider four case studies representing a large variety of population genetics investigations differing in their sampling strategies (noninvasive or traditional), in the type of organism studied (plant or animal) and the molecular markers used [microsatellites or amplified fragment length polymorphisms (AFLPs)]. In these data sets, the estimated genotyping error rate ranges from 0.8% for microsatellite loci from bear tissues to 2.6% for AFLP loci from dwarf birch leaves. Main sources of errors were allelic dropouts for microsatellites and differences in peak intensities for AFLPs, but in both cases human factors were non-negligible error generators. Therefore, tracking genotyping errors and identifying their causes are necessary to clean up the data sets and validate the final results according to the precision required. In addition, we propose the outline of a protocol designed to limit and quantify genotyping errors at each step of the genotyping process. In particular, we recommend (i) several efficient precautions to prevent contaminations and technical artefacts; (ii) systematic use of blind samples and automation; (iii) experience and rigor for laboratory work and scoring; and (iv) systematic reporting of the error rate in population genetics studies.

/pdf/how-to-track-and-assess-genotyping-errors-in-population-1ccjofq7qv.pdf

How to track and assess genotyping errors in population genetics studies.

Although genotyping errors affect most data and can markedly influence the biological conclusions of a study, they are too often neglected. Errors have various causes, but their occurrence and effect can be limited by considering these causes in the production and analysis of the data. Procedures that have been developed for dealing with errors in linkage studies, forensic analyses and non-invasive genotyping should be applied more broadly to any genetic study. We propose a protocol for estimating error rates and recommend that these measures be systemically reported to attest the reliability of published genotyping studies.

Genotyping errors: causes, consequences and solutions.

Amplified fragment length polymorphisms (AFLPs) are polymerase chain reaction (PCR)-based markers for the rapid screening of genetic diversity. AFLP methods rapidly generate hundreds of highly replicable markers from DNA of any organism; thus, they allow high-resolution genotyping of fingerprinting quality. The time and cost efficiency, replicability and resolution of AFLPs are superior or equal to those of other markers [allozymes, random amplified polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP), microsatellites], except that AFLP methods primarily generate dominant rather than co-dominant markers. Because of their high replicability and ease of use, AFLP markers have emerged as a major new type of genetic marker with broad application in systematics, pathotyping, population genetics, DNA fingerprinting and quantitative trait loci (QTL) mapping.

/pdf/aflp-genotyping-and-fingerprinting-4sw6vfeake.pdf

AFLP genotyping and fingerprinting

http://www.bio-nica.info/Biblioteca/Meudt2007AFLPApplicationAnalysis.pdf

Almost forgotten or latest practice? AFLP applications, analyses and advances.

Random amplified polymorphic DNA (RAPD) markers have been used for numerous applications in plant molecular genetics research despite having disadvantages of poor reproducibility and not generally being associated with gene regions. A novel method for generating plant DNA markers was developed based on the short conserved region flanking the ATG start codon in plant genes. This method uses single 18-mer primers in single primer polymerase chain reaction (PCR) and an annealing temperature of 50°C. PCR amplicons are resolved using standard agarose gel electrophoresis. This method was validated in rice using a genetically diverse set of genotypes and a backcross population. Reproducibility was evaluated by using duplicate samples and conducting PCR on different days. Start codon targeted (SCoT) markers were generally reproducible but exceptions indicated that primer length and annealing temperature are not the sole factors determining reproducibility. SCoT marker PCR amplification profiles indicated dominant marker like RAPD markers. We propose that this method could be used in conjunction with these markers for applications such as genetic analysis, bulked segregant analysis, and quantitative trait loci mapping, especially in laboratories with a preference for agarose gel electrophoresis.

Start Codon Targeted (SCoT) Polymorphism: A Simple, Novel DNA Marker Technique for Generating Gene-Targeted Markers in Plants

A number of PCR-based techniques can be used to detect polymorphisms in plants. For their wide-scale usage in germplasm characterisation and breeding it is important that these marker technologies can be exchanged between laboratories, which in turn requires that they can be standardised to yield reproducible results, so that direct collation and comparison of the data are possible. This article describes a network experiment involving several European laboratories, in which the reproducibility of three popular molecular marker techniques was examined: random-amplified fragment length polymorphism (RAPD), amplified fragment length polymorphism (AFLP) and sequence-tagged microsatellites (SSR). For each technique, an optimal system was chosen, which had been standardised and routinely used by one laboratory. This system (genetic screening package) was distributed to different participating laboratories in the network and the results obtained compared with those of the original sender. Different experiences were gained in this exchange experiment with the different techniques. RAPDs proved difficult to reproduce. For AFLPs, a single-band difference was observed in one track, whilst SSR alleles were amplified by all laboratories, but small differences in their sizing were obtained.

Reproducibility testing of RAPD, AFLP and SSR markers in plants by a network of European laboratories

For the application of molecular techniques to wide-scale diversity screening it is important that the different screening techniques employed can be standardized to yield reproducible results across laboratories in order that direct collation and comparison of the data are possible (1). RAPDs (Random Amplified Polymorphic DNA) involve the use of a single ‘arbitrary’ primer (purchasable from commercial companies) in a PCR reaction and results in the amplification of several discrete DNA products (see Chapter 9). It is now widely recognized that to obtain reproducible band profiles on the gels it is absolutely essential to maintain consistent reaction conditions. Numerous studies have reported the separate effects of altering different parameters, such as ratio of template DNA/primers, concentration of Taq polymerase and Mg concentration, on the bands obtained (2–5). A network reproducibility test was carried by nine European laboratories (L1-L9) to determine whether RAPD profiles could be reproduced by different laboratories if all details of the reaction conditions were standardized (1).

R. L. Aert

Papers

Reproducibility testing of RAPD, AFLP and SSR markers in plants by a network of European laboratories

Reproducibility testing of RAPDs by a network of European laboratories