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. 2020 Oct 15;11:595902. doi: 10.3389/fmicb.2020.595902

FIGURE 1.

FIGURE 1

Protocol schema and representative images of four cervical cytological grades used in the study. (A) Sample collection, DNA extraction, HPV genotyping by Sanger sequencing and genomic CpG profiling of loci-specific promoters by PSQ. Sequencing results are used for statistical modeling, prediction and classification. (B) Four categories of liquid-based cervical cytology: NILM, ASC-US, LSIL, and HSIL with cytomorphologic features of disease progression, i.e., increased nuclear enlargement, nuclear membrane irregularity, nuclear/cytoplasmic ratio, and chromatin coarseness (ThinPrep Pap smear, 50x magnification). Bottom, binarized classification of 4 cytological grades used as outcomes (“0” and “1”) for logistic regression. Three distinct, sequential logit models were used for outcome prediction by molecular signatures. (C) PyroMark Q48 PSQ instrument and 48-well sample disk (expanded) used for DNA methylation analysis. Bottom, the PSQ CpG assays for three host genes: ADCY8, CDH8, and ZNF582 are shown according to chromosomal locations (red line). The representative pyrograms with assay specific CpG sites (blue-gray columns) are shown with sequence specific, light-intensity peaks along the x- and y-axis, respectively. Chromosome ideograms adapted from NCBI Map Viewer (www.ncbi.nlm.nih.gov/genome/guide/human)]. ASC-US, atypical squamous cells of undetermined significance; chr, chromosome; gDNA, genomic DNA; HSIL, high-grade squamous intraepithelial lesion; LSIL, low-grade squamous intraepithelial lesion; NILM, negative for intraepithelial lesion or malignancy; PCR, polymerase chain reaction; PSQ, pyrosequencing. Photo credit (cytology): Bradie Bishop, MD.