Generating genomic platforms to study Candida albicans pathogenesis

Mélanie Legrand; Sophie Bachellier-Bassi; Keunsook K. Lee; Yogesh Chaudhari; Hélène Tournu; Laurence Arbogast; Hélène Boyer; Murielle Chauvel; Vitor Cabral; Corinne Maufrais; Audrey Nesseir; Irena Maslanka; Emmanuelle Permal; Tristan Rossignol; Louise A. Walker; Ute Zeidler; Sadri Znaidi; Floris Schoeters; Charlotte Majgier; Renaud A. Julien; Laurence Ma; Magali Tichit; Christiane Bouchier; Patrick Van Dijck; Carol A. Munro; Christophe d’Enfert

Sanger sequencing of both 5' and 3' ends was performed to confirm ORF identity and exclude clones containing primer or recombination errors. Previous studies have reported mutation rates in primers of 3-10% (26, 29) . Similar rates were observed in this work. Unlike other Gatewayâ„¢ recombinational cloning projects (15, 29) , we did not see major differences in cloning efficiency for ORFs up to 4 kb. A decrease in success rate was observed only for ORFs >4 kb. This size bias could be attributed to an increased difficulty to amplify the ORF by PCR, a reduced efficiency of the Gatewayâ„¢ BP Clonaseâ„¢ reactions with long PCR products, and the error rate of the PCR polymerase, which increases with longer products. In addition, we also corrected the sequences of 110 ORFs with sequence ambiguities (N-tracts and IUPAC code nucleotides) according to the Candida Genome Database and identified 116