1. High-Throughput and Sensitive Quantification of Circulating Tumor DNA by Microfluidic- Based Multiplex PCR and Next-Generation Sequencing 
Yinghui Guan, Oleg Mayba, Thomas Sandmann, Shan Lu, Younjeong Choi, Walter C. Darbonne, Vincent Leveque, Lisa Ryner, Eric Humke, Nga W.R. Tam, Sundari Sujathasarma, Anna Cheung, Richard Bourgon, Mark R. Lackner, Yulei Wang 
The Journal of Molecular Diagnostics 
Volume 19, Issue 6, Pages (November 2017)
DOI: /j.jmoldx
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

2. Figure 1
PreAmp MMP-Seq work flow for ctDNA. This is a schematic illustration showing a work flow for a ctDNA panel. Plasma DNA (5 ng) is first preamplified with 96 to 121 multiplex PCR assays, followed by multiplex target enrichment in Fluidigm Access Array, as described previously.19 The libraries were then sequenced on a MiSeq instrument. BC, barcode sequence; cfDNA, cell-free DNA; CS, Fluidigm common sequence; F, forward primer; PE, Illumina sequencing paired-end sequence; R, reverse primer; TS, target specific.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

3. Figure 2
Performance assessment of PreAmp MMP-Seq work flow. A: Latin square (LSQ) control DNA samples are run with the PreAmp and regular MMP-Seq work flows on a ctDNA panel to evaluate amplicon coverage. B: Expected mutations from LSQ samples detected with no PreAmp and PreAmp MMP-Seq protocols are plotted to compare sensitivities of the two protocols. C: Allele fractions (AFs) of variant calls from two replicate PreAmp MMP-Seq runs are compared to evaluate reproducibility. The dashed lines in B and C mark the 0 point of AFs on the x and y axes. D: Linearity of four expected mutations in LSQ samples detected by the PreAmp protocol. Solid lines represent linear regression trend line, and dotted lines on each side represent 95% CIs. E: There are 142 engineered hotspot mutations in the AcroMetrix Oncology Hotspot Control (AOHC) that are covered by ctDNA panel amplicons. The distributions of allele frequencies of the 142 hotspots are plotted in histograms. Separate panels represent data from different titrations of AOHC samples. The expected average frequency measured independently by the vendor through next-generation sequencing is indicated on top of each panel of histograms. The x axis is shown on a log2 scale. The expected AFs of serially diluted AOHC samples are highlighted with dashed lines in each histogram.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

4. Figure 3
Determination of position/substitution-specific errors with the empirical bayesian error model. Forty-three normal plasma samples were profiled with the ctDNA panel using the PreAmp MMP-Seq. A: Error rate distribution of all ctDNA amplicons. B: Error rate distribution of ctDNA amplicons labeled by reference (Ref) bases. C: Error distribution of amplicon HRAS p.Q61H_1 (Supplemental Table S2) separated by all potential base substitution types. Only nonzero observed error rates are used for visualization. D: Density plot of estimated error distribution, as shown in a black line, and observed error rates (red ticks at the bottom) are shown for two representative positions within the ctDNA panel of amplicons. Chr, chromosome; MAD, median absolute deviation.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

5. Figure 4
Amplicon coverage and error rates of tissue hotspots in normal plasma. Longitudinal plasma samples from φ DMUC5754A trials were profiled using the ctDNA PreAmp MMP-Seq. A: Average sequencing coverage per amplicon in 81 plasma samples. B: Error rates and their 95% CIs (error bars) calculated on the basis of an empirical bayesian model are plotted for 29 positions where clinically relevant variants were detected in matched formalin-fixed, paraffin-embedded tissues. Solid lines indicate the average, and the dashed line in B shows the median.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

6. Figure 5
Clinical utilities of ctDNA work flow on monitoring disease burden. Longitudinal levels of mutations found in both plasma and matched tissues from four patients are shown. The ctDNA levels (pg/mL plasma) were calculated by multiplying the frequency of a mutation with the mass (pg) of cell-free DNA yield/mL plasma. The levels of ctDNA at certain treatment time points are compared with those at baseline before treatment. The y axes of all four subplots are percentage change from baseline. Percentage change in longitudinal HE4 or CA-19-9 levels and tumor size are also plotted in each graph for comparison. Also, 40% HE4 and 50% CA-19-9 reductions are associated with clinical response in DMUC5754A and are shown in a dashed line for each patient. SLD, size at the longest dimension.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions