Article Figures & Data
Figures
- FIG. 1.
Simulation results of 10 randomly selected S gene reference sets, each representing all HBV genotypes. The x axis displays the groups of all four reactions (TF, TR, CF, and CR, where “F” is “forward” and “R” is “reverse”) and any combination of three cleavage reactions, two reactions, or one reaction. The y axis displays the average percentage of sequences falling into the individual power of discrimination categories (very weak, no fill; weak, light gray; medium, dark gray; and strong, black). For example, if all four reactions were used, 100% of the data are discriminated with strong power, if only the CF reaction was used, approximately 38% were discriminated with strong power, etc. A threshold of 2 (the equivalent of two full peak changes) is required to distinguish one sequence from another with good confidence.
- FIG. 2.
Pilot experiment data. (A) Box plots of the cumulative scores from all four reactions. The groups are PCR-positive specimens with four successful reactions (PCRpos; n = 69), positives with at least one failed reaction (pos*; n = 15), negative controls (neg; n = 12), and an irrelevant group consisting of HBV core gene sequences (irrelevant; n = 240). The ordinate displays the score values. The box represents the first through the third quartile of the data, the median is denoted with a “−” sign, and the whiskers spread to the minimum and the maximum values. A dotted line is drawn at the genotype cutoff value of 0.6. (B) Score data from the 15 specimens of the pilot experiment that had at least one reaction failure (pos*). The scores were calculated after analysis of all four mononucleotide reactions, TF, TR, CF, and CR separately (open shapes). The black circles are the cumulative scores acquired using all four reactions and the same reference set. Each vertical gridline contains all five data scores (TF, TR, CF, CR, and cumulative) from one specimen. Dotted line denotes the genotype cutoff value of 0.6. All values below the cutoff represent individual failed nucleotide reactions.
- FIG. 3.
Matching score values and corresponding viral titers. Shown is a scatter plot of all MS data from all PCR-positive specimens (n = 1,121), including duplicates. The scores indicate the matching of a particular specimen to the closest member of the reference set (n = 153). The dotted line denotes the genotype cutoff score (0.6).
- FIG. 4.
Box plots showing the effect of PCR quality and the combination of cleavage reactions on score values. The dotted line is placed at the genotype cutoff score (0.6). Score values are on the ordinates. (A) Post-PCR product quality. Scores are grouped as follows by the number of successful reactions per sample: samples with all four successful reactions (n = 591), samples with three successful reactions (n = 139), samples with two successful reactions (n = 36), and samples with one successful reaction (n = 16). (B) PCR quality. Shown is a comparison between the scores of fresh PCR products and scores of PCR products stored over 2 months.
- FIG. 5.
Effects of the complexity of the reference set on the score. Box plot of the MS data from all cases analyzed (n = 756) using a minimal reference set (n = 15), the large reference database (n = 153), and an enhanced reference set that is enriched with the novel sequences generated automatically during the iSEQ analysis (n = 220).
- FIG. 6.
Effects of the genotype and viral complexity on the score. (A) Box plot of the score data grouped by genotype. Data from PCR-positive specimens (n = 756) were analyzed using a large reference set (n = 153). The genotypes are indicated on the abscissa. The dotted line is placed at the genotype call cutoff score (0.6). E* indicates improved scoring of genotype E specimens after analysis with the enriched reference set (n = 220). (B) Box plot of the scores of the acute specimens in comparison to the scores from the chronic specimens analyzed against two different reference sets, large (n = 153) and enriched (n = 220).
Tables
- TABLE 1.
Inter- and intragenotype divergence among the 153 sequences of the large reference seta
Genotype % nucleotide divergencea A B C D E F G H A 0.2-5.0 3.7-8.1 3.8-8.8 3.7-7.5 4.0-7.5 5.7-9.4 2.8-5.3 5.4-8.6 B 0.2-3.5 4.2-8.1 4.2-7.2 4.3-6.3 4.9-7.7 5.9-7.2 5.0-6.6 C 0.2-4.3 4.4-7.9 4.7-6.8 4.7-7.2 4.2-7.4 5.0-7.7 D 0.2-3.3 3.2-5.4 4.5-7.4 4.2-5.6 4.9-6.4 E 0.2-0.7 4.9-6.5 4.3-4.9 5.2-5.7 F 0.2-2.8 5.4-6.8 2.3-3.5 G 0.2-0.8 6.1-6.4 H 0.2-0.4 ↵ a The numbers represent the percentages of nucleotide divergence between and within the genotypes based on a 441-bp fragment of the HBV S gene.
