Plasmid-Based Materials as Multiplex Quality Controls and Calibrators for Clinical Next- Generation Sequencing Assays  David J. Sims, Robin D. Harrington, Eric C. Polley, Thomas D. Forbes, Michele G. Mehaffey, Paul M. McGregor, Corinne E. Camalier, Kneshay N. Harper, Courtney H. Bouk, Biswajit Das, Barbara A. Conley, James H. Doroshow, P. Mickey Williams, Chih-Jian Lih  The Journal of Molecular Diagnostics  Volume 18, Issue 3, Pages 336-349 (May 2016) DOI: 10.1016/j.jmoldx.2015.11.008 Copyright © 2016 Terms and Conditions

Figure 1 Design of control plasmids. A: Map of a representative control plasmid, pNF1_34041. Each control plasmid was constructed by inserting approximately 1000 bp of genomic DNA (blue box) spanning the mutation of interest (MOI) (red star). A 6-bp (ACATCG) molecular barcode (orange rectangle in B) was inserted near the MOI to track variant reads. Single-cut restriction sites are indicated by yellow triangles. B: Coordination of the molecular barcode with the MOI. A subset of sequencing reads from MOIs [an A deletion (red box)] and 6-bp molecular barcode confirms the mutation is plasmid borne. The Journal of Molecular Diagnostics 2016 18, 336-349DOI: (10.1016/j.jmoldx.2015.11.008) Copyright © 2016 Terms and Conditions

Figure 2 Control plasmid spiked-in genome (CPSG) titration workflow. Schematic showing the procedure for generating a CPSG sample. Briefly, plasmids are linearized, quantified, pooled, and spiked into a background genome at a determined copy number ratio. The Journal of Molecular Diagnostics 2016 18, 336-349DOI: (10.1016/j.jmoldx.2015.11.008) Copyright © 2016 Terms and Conditions

Figure 3 Parallel spike-in study strategy. A: Schematic diagram of parallel spike-in study. A pair of serially diluted sample sets made by spiking control plasmid pBRAF_476 or genomic DNA from cell line MALME-3M carrying the same BRAF V600E mutation, into the hapmap genomic DNA at the same titration points, followed by sequencing and regression analysis of the expected allele frequency versus the observed allele frequency. B: Scatterplot and regression analysis for pBRAF_476/MALME-3M and pAPC_18584/HCT-15. Linear regression models were fit for each. FFPE, formalin-fixed, paraffin-embedded; PGM, Personal Genome Machine; VAF, variant allele frequency. The Journal of Molecular Diagnostics 2016 18, 336-349DOI: (10.1016/j.jmoldx.2015.11.008) Copyright © 2016 Terms and Conditions

Figure 4 CPSG13 reproducibility during 18 months with three operators. A: CPSG13 was repeatedly sequenced by the National Cancer Institute's MPACT (NCI-MPACT) assay 90 times by three operators (OP1, OP2, and OP3) during an 18-month period. Boxplots showing the variant allele frequency (VAF) distribution binned by plasmid for each operator were plotted. The dashed line represents the 25% VAF point at which the plasmids were intended to be titrated. B: The mean VAF for each of the 13 plasmids and three operators was plotted as a green solid line during 18 months to evaluate the variability and stability of the material. The blue dashed line represents the expected 25% VAF. The red line represents a regression model through the series. The gray shadow represents the range of VAF for the 13 plasmids at each test date on the x axis. The Journal of Molecular Diagnostics 2016 18, 336-349DOI: (10.1016/j.jmoldx.2015.11.008) Copyright © 2016 Terms and Conditions

Figure 5 Comparison of the three CPSG51 data analysis pipelines. CPSG51 was sequenced by the National Cancer Institute's MPACT assay, and the same raw data were analyzed by Torrent Suite Software (TSS) versions 3.2.1, 4.0.2, and 4.4.2. Tile plots indicating whether a variant was detected (red box) or not (gray box) were generated for each of the three pipeline versions. Each row represents a mutation in a control plasmid, and each column represents a titration point. The plasmid name and the variant type (in parenthesis) are indicated on the left. HP, homopolymeric region; indel, insertion/deletion; SNV, single-nucleotide variant. The Journal of Molecular Diagnostics 2016 18, 336-349DOI: (10.1016/j.jmoldx.2015.11.008) Copyright © 2016 Terms and Conditions

Figure 6 Comparison of the three CPSG51 assay platforms. A 32-plasmid subset detectable across three different next-generation sequencing chemistries and platforms was compared. A tile plot was generated for each of the three platforms, and an indication was made as to whether the variant was detected (red box) or not (gray box). Each row represents a mutation in a control plasmid, and each column represents a titration point. The plasmid name and the variant type (in parenthesis) are indicated on the left. HP, homopolymeric region; indel, insertion/deletion; TSCA, TruSeq Custom Amplicon; SNV, single-nucleotide variant; WES, whole exome sequencing. The Journal of Molecular Diagnostics 2016 18, 336-349DOI: (10.1016/j.jmoldx.2015.11.008) Copyright © 2016 Terms and Conditions