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    ries, Inc., Hercules, CA). For a subset of targets, TaqMan Gene Expression Assays (Thermo Fisher Scientific) were used according to the manufacturer's instructions on an Applied Biosystems™ 7500 real-time PCR system. For fusions that did not have a commercial qPCR assay available, a hydrolysis probe was designed to span the specific breakpoint, and a qPCR assay was performed in conjunction with the gene-specific primers associated with the panel to confirm fusion presence (95°C for 5 minutes, 40 cycles: 95°C for 5 seconds, 60°C for 1 minute).
    An Integrated NGS Workflow Enabling Streamlined Analysis of DNA and RNA
    An overview of the integrated targeted NGS workflow for DNA and RNA is shown in Figure 1. This approach enables the interrogation of both DNA and RNA through a single isolation of TNA from low-quality or low-quantity FFPE or FNA specimens. In order to determine the suitability of the TNA for NGS, a preanalytical QC assay is performed for both DNA and RNA with a readout of amplifiable template molecules for each analyte. Library preparation employs a two-pool strategy that utilizes a two-step PCR with shared CT-99021 and cycling conditions across the DNA and RNA libraries to streamline the workflow. Postsequencing, DNA and RNA library pools are demultiplexed and analyzed through separately optimized informatics pipelines, the results of which are integrated together to assess implications across both categories of nucleic acid. The combined workflow detects and quantifies SNVs, INDELs, CNVs, fusions, splice variants, and expression of select genes to minimize cost and specimen consumption. To demonstrate the clinical research value of this integrated NGS method, we designed a panel (Table 2) for NSCLC that covers DNA mutation hotspots and RNA fusions, splice variants, and expression targets. Panel content was selected based on a review of the NCCN guidelines, COSMIC database, TCGA results, and active clinical trial research (see Methods).
    Preanalytical QC Analysis of DNA and RNA
    We applied the preanalytical QC assays to a cohort of 219 NSCLC patients (Table 1; Supplemental Table S1) to quantify the amplifiable DNA and RNA fractions from TNA isolations (Supplemental Table S2). The cohort consisted of 109 FFPE CNB specimens and 110 FFPE surgical resections representing 121 LUADs and 98 LUSCs.
    Figure 1. Overview of the NGS procedure targeting both DNA and RNA. An integrated NGS workflow enables parallel analysis of DNA and RNA through a real-time qPCR QC analysis and target enrichment through two-step PCR.
    840 An Integrated Next-Generation Sequencing System Haynes et al. Translational Oncology Vol. 12, No. 6, 2019
    Figure 2. Amplifiable DNA and RNA yields are correlated and predictive of sequencing quality. (A) Assessment of CNBs (purple) and surgical resections (green) reveals a relationship between amplifiable DNA and RNA copies/μl as determined by RT-qPCR. (B) DNA-Seq and (C) RNA-Seq passing filter (PF) mapping rates to panel targets (y-axis) are predicted by amplifiable template molecules used for library prep (x-axis). Functional QC thresholds are shown as dashed vertical lines in panels B and C.
    The number of amplifiable DNA copies from these FFPE samples was significantly correlated with amplifiable RNA copies as defined by the RNA endogenous control target [Spearman's correlation coefficient (SCC): 0.81, P b2.2e-16, Figure 2A]. On average, surgically resected FFPEs yielded more DNA and RNA than CNBs (Figure 2A). By utilizing the matched DNA copy number data as a reference to determine the functional cell count equivalent for a given specimen (assuming diploidy of the DNA control), we calculated that the RT-qPCR endogenous control RNA target was expressed at approximately 2.8 copies per cell on average across all specimens, which are indicative of stable expression in the same copy number range as our DNA control.
    We defined QC thresholds for DNA libraries through independent studies [27] and established 200 amplifiable copies as the minimum input required to reliably call DNA variants down to 5% VAF while avoiding false-negative calls. A similar approach was taken for RNA libraries [28], and a minimum functional cDNA copy input of 200 copies was established as the requirement to reliably call fusion variants down to 5% cell positivity. Based on these criteria, we found
    Library preparation and analysis were performed on all specimens independent of preanalytical QC results. Consistent with other studies [25,26], the number of functional template molecules used in preparation of NGS libraries was significantly associated with the percentage of reads mapping to the intended targets for both targeted DNA-Seq and RNA-Seq targets (SCC: 0.87 and 0.75 for DNA and RNA, respectively; Fig. 2, B and C).