Genechip resequencing assay kit

Amplified DNA was fragmented and labeled using the GeneChip Resequencing Assay Kit (Affymetrix Inc., Santa Clara, CA). After overnight hybridization at 45°C, the RMA was washed, stained and scanned according to Affymetrix’ instructions. The raw image file (.DAT), obtained after the scan, was converted to a fluorescence intensity file (.CEL). Bases were called by the GeneChip Sequence Analysis Software (GSEQ v 4.1) which uses a derivative of the ABACUS base-calling algorithm [20] (link). This algorithm consists of an automated statistical method that analyses raw RMA hybridization data and optimizes the base-calling process. For each base-call, a quality score is calculated from the difference between the best fitting model and the second best one. If this score is below a chosen threshold, an undetermined base “N” is assigned to that position. Sequences were output in FASTA format; for each HPV sequence obtained, the call rate value was calculated as the ratio between the number of bases determined (A or T or C or G) and the entire sequence length. The accuracy of the RMA process was determined as the ratio between the number of correctly determined bases and the total number of determined bases, by comparison with the known sequence of the strains tested.

Analysis of mtDNA sequence variations was done using GeneChip™ Human Mitochondrial Resequencing Arrays 2.0 (MitoChip v2.0) [44 (link)]. For processing of the arrays, the GeneChip® CustomSeq® Resquencing Array Protocol version 2.1 was used with the following modification: instead of using long-range PCR, mtDNA was amplified using the REPLI-g mitochondrial DNA kit (Qiagen Hilden, Germany), as it has been described for MitoChip experiments before [45 (link)], and purified using Agencourt® AMPure® XP magnetic beads (Qiagen Supplementary Protocol, REPLI-g mitochondrial DNA kit). After purification, 270 ng of mtDNA was fragmented and labeled using the GeneChip® Resequencing Assay Kit (Affymetrix, now part of ThermoFisher Scientific). After hybridization, MitoChip v2.0 arrays were washed and stained in a Fluidics Station 450 before being scanned in Affymetrix GeneChip Scanner 3000 7G. CEL files were acquired by GeneChip® Operating Software 1.4 (GCOS 1.4.0.036) and analyzed with GeneChip® Sequence Analysis Software (GSEQ 4.1). Complete microarray data are available at Gene Expression Omnibus (GEO accession number: GSE211250).