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

Validation of entry clones by DNA sequencing and bioinformatics analysis. Sanger sequencing of the 5'-end of each BP clone was performed with the 207VER-F oligonucleotide (see Supplemental Table 2 for oligonucleotide sequences except those used to amplify ORFs) to confirm that the expected ORF had been cloned. The 3'-ends were also sequenced with the 207VER-R oligonucleotide to ensure that the oligonucleotides did not carry deletions. All inserts validated by Sanger sequencing were systematically subjected to full-length sequence analysis using Illumina technology as described in Chauvel et al. (11) . If nonsense or frameshift mutations were observed in a cloned ORF, another colony was checked, or the ORF reamplified and cloned again. If these attempts were unsuccessful, cloning of the ORF was abandoned. Clones containing contiguous deletions or insertions of multiples of 3 bp were accepted. Missense mutations and those located within introns were accepted. It should be noted that further analysis of each mutation-containing ORF is required to determine whether or not mutations affect the function of the ORF.