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Open Access Highly Accessed Methodology article

A 454 multiplex sequencing method for rapid and reliable genotyping of highly polymorphic genes in large-scale studies

Maxime Galan1*, Emmanuel Guivier1, Gilles Caraux23, Nathalie Charbonnel1 and Jean-François Cosson1

Author Affiliations

1 INRA EFPA, UMR CBGP (INRA/IRD/Cirad/Montpellier SupAgro), Campus international de Baillarguet, CS 30016, F-34988 Montferrier-sur-Lez cedex, France

2 Laboratoire d'Informatique de Robotique et de Microélectronique de Montpellier (LIRMM) CNRS: UMR5506 - Université Montpellier II - Sciences et Techniques du Languedoc, France

3 Montpellier SupAgro, 2 Place Pierre Viala, F-34060 Montpellier, France

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BMC Genomics 2010, 11:296  doi:10.1186/1471-2164-11-296

Published: 11 May 2010

Additional files

Additional file 1:

Additional Information. Probabilities of observing artifactual sequences by substitution errors. Probability f(r,m,n) of observing at least r sequences of each of the m variants potentially observed for the n sequences of a given sample.

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Additional file 2:

Fig S1, Fig S2, Fig S3, Fig S6. Figure S1. Histograms showing the number of sequences obtained for individual variants (Nj) for pools A and B. Insets display distributions for values below fifty. Figure S2. Histograms of the number of sequences obtained for each sample (Ni) after the first step of data processing for pools A and B. Figure S3. Confidence level for genotyping. f is the probability of amplifying, at least r times, all the different variants of the gene studied for a given sample. This probability depends on n, the total number of sequences by sample, and m, the maximal number of variants for the gene within a sample. T1 is the threshold value that corresponds to the minimal number of sequences required per individual to determine its complete genotype, with a 10-3 probability of missing variants. Plots are given for different values of r = 1, 2, 5 and 10. Figure S6. The number of sequences obtained for each forward and reverse tag after the first step of data processing for pools A and B.

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Additional file 3:

Fig S4, Fig S5. Figure S4. Histograms showing the distributions of Fij, the frequency of each variant j within each individual sample i. Data were grouped as a function of rodent genera. m is the maximal number of variants for the gene within a sample. Figure S5. True and artifactual variants of DRB exon 2 for a black rat (Rattus rattus). The two variants validated by our data processing are shown in green and blue, respectively. We obtained 36 sequences for the variant highlighted in green and 26 for the blue variant. Other variants are artifactual (n = 20). Variants corresponding to a mixture of blue and green correspond to recombinant chimeric sequences derived from a mixture of sequences of DRB*003 and DRB*006. Other artifactual variants corresponded to substitutions (sites in red, DRB*532, DRB*926, DRB*438, DRB*1412 and DRB*1636), indel (DRB*1653), pseudogenes orthologous to RT1-Hb (DRB*462) and the paralog DQB (DRB*1008).

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