Sequences and PCR parameters for our 2-color fluorometric VHSv assay.

<p>Primers, probes, internal standards (IS), and synthetic templates are specified. F = forward primer, R = reverse primer, NT = native template. <i>Italics</i> = modified nucleotides in NT probe.</p

Relationship between the numbers of observed versus expected molecules when NT:IS concentrations are 1∶1.

<p>Results are based on dilutions of the Native Template (NT) and Internal Standard (IS) of 6×10⁶, 6×10⁵, 6×10⁴, 6×10³, 6×10², 60, 6, and 0.6 molecules for VHSv and <i>actb1</i>. The 2-color fluorometric assay yields a linear relationship for (a) VHSv over seven orders of magnitude (from 6×10⁶ to 6×10⁰ VHSv molecules) with a slope of 1.00 (<i>R</i>² = 0.99, <i>F = </i>9404.00, df = 1, 5, <i>p</i><0.001), and mean CV of 9%. A linear trend also is obtained for (b) <i>actb1</i> (<i>R</i>² = 0.99, <i>F = </i>1347.00, df = 1, 5, <i>p</i><0.001). Slope = 1.04, mean CV = 10%. Error bars = standard error of triplicate runs.</p

Specificity of the 2-color fluorometric test.

<p>– = negative result (no amplification), + = positive result.</p><p>Isolates obtained from:</p>a<p>Western Fisheries Research Center, USGS, Seattle, WA, USA.</p>b<p>Cornell University College of Veterinary Medicine, Ithaca, NY, USA.</p>c<p>Finnish Food Safety Authority Evira, Finland.</p>d<p>Universidad de Santiago de Compostela, Spain.</p>e<p>Fisheries and Oceans Canada, Pacific Biological Station, BC, Canada.</p

Mean log numbers of VHSv molecules/10⁶<i>actb1</i> molecules from our new 2-color fluorometric assay versus the prior Agilent capillary electrophoresis approach.

<p>Results indicate a linear relationship between the two tests (<i>R</i>² = 0.91, df = 1, 38, <i>F = </i>396.40, <i>p</i><0.001) and do not significantly differ (<i>t = </i>1.42, df = 78, NS). Arrow = Estimated threshold concentration of VHSv for fish with clinical signs of infection using our new assay, from a <i>χ</i>² test of nine symptomatic fish (1×10³ VHSv molecules/10⁶<i>actb1</i> molecules = 3.6×10² VHSv molecules). Triangle = false negative range for SYBR® green qRT-PCR (1.0×10⁰–1.6×10² VHSv/10⁶<i>actb1</i> molecules = 0.6×10⁰–2.5×10² VHSv molecules). Square = false negative range for cell culture (1.0×10⁰–2.2×10³ VHSv/10⁶<i>actb1</i> molecules = 0.6×10⁰–6.1×10³ VHSv molecules.</p

Relationship between the number of observed and expected molecules for NT:IS concentrations of 1∶1–1∶20.

<p>The concentration of Native Template (NT) is held constant and the Internal Standard (IS) varied for dilutions of: 1∶1 (6×10⁴ molecules), 1∶2, 1∶3, 1∶4, 1∶5, 1∶6, 1∶7, 1∶8, 1∶9, 1∶10, 1∶12, 1∶14, 1∶16, 1∶18, and 1∶20 (3×10³molecules). The 2-color fluorometric assay yields a linear relationship for (a) VHSv (<i>R</i>² = 0.99, <i>F = </i>1514.00, df = 1, 13, <i>p</i><0.001) with a mean CV of 5% for dilutions 1∶1–1∶10 and 7% for concentrations down to 1∶20, and for (b) <i>actb1</i> (<i>R</i>² = 0.99, <i>F = </i>1283.00, df = 1, 13, <i>p</i><0.001), CV = 5% and 7%. The same linear pattern is observed when the IS was held constant and NT varied for (c) VHSv (<i>R</i>² = 0.99, <i>F = </i>5124.00, df = 1, 13, <i>p</i><0.001), CV = 5% for 1∶1–1∶10 and 7% for dilutions down to 1∶20, and (d) <i>actb1</i> (<i>R</i>² = 0.99, <i>F = </i>2434.00, df = 1, 13, <i>p</i><0.001), CV = 3% and 6%. Error bars = standard error of results for triplicate runs. Dotted line = partition of dilutions from 1∶1–1∶10 (right) and 1∶12–1∶20 (left).</p

Real-time PCR amplification plots for various experiments.

<p>ABI 7500 real-time PCR results for (a) a true VHSv positive fish sample, (b) the relationship between the VHSv Native Template (NT) and Internal Standard (IS) with the NT held constant and the IS varied, (c) the relationship between VHSv NT and IS with the IS held constant and the NT varied, and (d) the relationship between VHSv NT:IS with concentrations held constant for dilutions of 1∶1–1∶20. Green = NT, red = IS, y = fluorescence of the reporter dye minus the baseline (Δ fluorescence), x = cycle threshold.</p

Concentrations for the 2-color fluorometric VHSv assay.

<p>Dilution mixtures (A–H) used for the Internal Standards Mixture (ISM) <i>actb1</i>/VHSv are given in units of 10^x M.</p

Relative numbers of VHSv positives and negatives from our 2-color fluorometric and capillary electrophoresis StaRT-PCR assays, which indicates identical numbers of positives and negatives.

<p>Compared to these tests, for 43 fishes (25 positives, 18 negatives (including 5 laboratory controls)), (a) SYBR® green has 44% false negative error (<i>χ</i>² = 5.67, df = 1, <i>p</i> = 0.02), and cell culture has 56% error (<i>χ</i>² = 9.36, df = 1, <i>p</i> = 0.002). For 63 fish samples (39 positives, 24 negatives (including 11 laboratory controls)), (b) SYBR® green qRT-PCR has 33% false negative error (<i>χ</i>² = 5.37, df = 1, <i>p</i> = 0.02), whereas the 2-color fluorometric and capillary electrophoresis tests show zero false negatives.</p

Targeted RNA-Sequencing with Competitive Multiplex-PCR Amplicon Libraries

<div><p>Whole transcriptome RNA-sequencing is a powerful tool, but is costly and yields complex data sets that limit its utility in molecular diagnostic testing. A targeted quantitative RNA-sequencing method that is reproducible and reduces the number of sequencing reads required to measure transcripts over the full range of expression would be better suited to diagnostic testing. Toward this goal, we developed a competitive multiplex PCR-based amplicon sequencing library preparation method that a) targets only the sequences of interest and b) controls for inter-target variation in PCR amplification during library preparation by measuring each transcript native template relative to a known number of synthetic competitive template internal standard copies. To determine the utility of this method, we intentionally selected PCR conditions that would cause transcript amplification products (amplicons) to converge toward equimolar concentrations (normalization) during library preparation. We then tested whether this approach would enable accurate and reproducible quantification of each transcript across multiple library preparations, and at the same time reduce (through normalization) total sequencing reads required for quantification of transcript targets across a large range of expression. We demonstrate excellent reproducibility (R² = 0.997) with 97% accuracy to detect 2-fold change using External RNA Controls Consortium (ERCC) reference materials; high inter-day, inter-site and inter-library concordance (R² = 0.97–0.99) using FDA Sequencing Quality Control (SEQC) reference materials; and cross-platform concordance with both TaqMan qPCR (R² = 0.96) and whole transcriptome RNA-sequencing following “traditional” library preparation using Illumina NGS kits (R² = 0.94). Using this method, sequencing reads required to accurately quantify more than 100 targeted transcripts expressed over a 10⁷-fold range was reduced more than 10,000-fold, from 2.3×10⁹ to 1.4×10⁵ sequencing reads. These studies demonstrate that the competitive multiplex-PCR amplicon library preparation method presented here provides the quality control, reproducibility, and reduced sequencing reads necessary for development and implementation of targeted quantitative RNA-sequencing biomarkers in molecular diagnostic testing.</p></div

Performance of competitive amplicon library preparation with ERCC Reference Materials.

<p><b>a)</b> Measured signal abundance of ERCC targets in samples A, B, C and D. X-axis units are derived from Ambion product literature for the known concentration of ERCC spike-in controls (n = 104). <b>b)</b> Difference plots of data in panel A ordered numerically by ERCC ID. Each ERCC target depicted was measured at least once in all four samples A–D. For purposes of clarity, ERCC-170 is highlighted orange in panels A and B (n = 104). <b>c)</b> Samples C and D represent a 3∶1 and 1∶3 mixture, respectively, of samples A and B. These ratios were used to calculate expected measurements for samples C and D (X-axis). Actual measurements of samples C and D are plotted on the Y-axis (n = 52). <b>d)</b> Coefficient of variation (CV) in measurements of ERCC targets in samples A-D, for those assays with at least two IS dilution points. Red line depicts expected CV based on a Poisson sampling (n = 95). <b>e)</b> ROC curves to detect fold change with corresponding area under the curve (AUC) with 95% confidence intervals. ROC curves are derived from the comparison of differential ratio subpools of ERCC targets in samples: A vs. B, A vs. C, A vs. D, B vs. C, B vs. D and C vs. D. Results for 1.1-fold change represent a range of differential ratio subpools [1.05–1.174] (controls n = 100, tests n = 96); 1.25-fold change [1.175–1.374] (controls n = 163, tests n = 163); 1.5-fold change [1.375–1.74] (controls n = 229, tests n = 227); 2.0-fold change [1.75–2.49] (controls n = 229, tests n = 223); ≥4.0-fold change [2.5–10.0] (controls n = 286, tests n = 290).</p