1. Multiplexed Reference Materials as Controls for Diagnostic Next-Generation Sequencing 
Emily M. Kudalkar, Naif A.M. Almontashiri, Catherine Huang, Bharathi Anekella, Mark Bowser, Elizabeth Hynes, Russell Garlick, Birgit H. Funke 
The Journal of Molecular Diagnostics 
Volume 18, Issue 6, Pages (November 2016)
DOI: /j.jmoldx
Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

2. Figure 1
Production of biosynthetic plasmids. Approximately 1000 bp surrounding each variant of interest (represented by white stars) was synthesized and cloned into a common plasmid backbone for all 10 variants. Left: The plasmids were quantified by ddPCR and pooled at equimolar ratios. The pooled plasmids were then spiked into reference genomic DNA (GM24385) at three ratios: 45%, 50%, and 55%. Right: The 10 constructs were cloned into one 10-kb plasmid and spiked into reference genomic DNA (GM24385) at a 50% ratio. ddPCR, droplet digital PCR.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

3. Figure 2
Expected versus observed allelic fractions. Allele frequency detected for all variants spiked into gDNA at 45%, 50%, 55%, and 50% for 10-kb plasmid. Mean allele frequency is calculated from the average of the replicates from four independent runs. Bonferroni correction was used to correct for multiple comparison. Data expressed as means ± SD. n = 4 independent runs, including 2 intrarun replicates for each variant. gDNA, genomic DNA; indel, insertion and/or deletion; Sub, substitution.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

4. Figure 3
Comparable detection of allele frequency between plasmid multiplexed controls and germline variants (patient genomic DNA). Data are expressed as means ± SD. n = 8 independent libraries (2 per run); n = 1 germline sample.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

5. Figure 4
Closeness to the target molecular ratios. Target allele fractions (0.45, 0.50, 0.55 for the pooled plasmid mix, 0.50 for the 10-kb plasmid) are indicated on the x axis. The differences of the observed AFs (▵) are plotted for all replicates (all variants were run in duplicate on four separate runs). SNVs and small indels (represented by p. Arg502Trp and p.Trp792Valfs) consistently yielded AFs close to the target AFs, whereas the larger indel (c _ del) shows both a higher deviation and variability. For the 10-kb plasmid run 4 represents an independently manufactured lot. n = 3 representative variants. AF, allelic fraction; indel, insertion and/or deletion; SNV, single nucleotide variant.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions