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Open Access Research article

MHC genotyping of non-model organisms using next-generation sequencing: a new methodology to deal with artefacts and allelic dropout

Simone Sommer1*, Alexandre Courtiol1 and Camila J Mazzoni12

Author Affiliations

1 Evolutionary Genetics, Leibniz-Institute for Zoo and Wildlife Research (IZW), Alfred-Kowalke-Straße 17, D-10315 Berlin, Germany

2 Berlin Center for Genomics in Biodiversity Research, Koenigin-Luise-Straße 6-8, D-14195 Berlin, Germany

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BMC Genomics 2013, 14:542  doi:10.1186/1471-2164-14-542

Published: 9 August 2013

Additional files

Additional file 1: Figure S1:

Describes the fusion primers and barcode combinations for 454 library preparation. Figure S2 contains an alignment of amino acid sequences of D. sublineatus MHC class II DRB alleles detected by cloning/Sanger sequencing and/or 454 pyrosequencing and outlines antigen-binding sites [43]. Figure S3 shows the allele frequencies in individuals genotyped by cloning/Sanger sequencing and 454 pyrosequencing. Figure S4 indicates the comparison of levels of individual MHC class II DRB diversity obtained by conventional cloning/Sanger sequencing and next-generation 454 pyrosequencing. Figure S5 outlines the predicted minimum number of reads (T1Min Amp Eff) required to determine a complete genotype for at least two reads per allele (99.9% confidence level). Figure S6 shows the same as Figure S5 but for at least three reads per allele.

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Additional file 2: Table S1:

Shows the MHC-DRB diversity in D. sublineatus detected by cloning/Sanger sequencing and 454 pyrosequencing using the identical individuals. Table S2 shows a list of the standardised allele amplification efficiencies. Table S3 shows the predicted minimum number of reads necessary to obtain at least two reads per allele based on minimum amplification efficiency. Table S4 shows the predicted minimum number of reads necessary to obtain at least three reads per allele based on minimum amplification efficiency.

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Additional file 3: Text S1:

Describes possible adjustments of the reads filtering pathway (Figure 1) and our allele and artefact identification workflow (Figure 2) to different needs in forthcoming MHC studies.

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Additional file 4:

The R-Codes used to estimate the allele’s amplification efficiency and the different T1 thresholds.

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