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Table 2 Overview of study objectives and strategy

From: Evaluation of an integrated clinical workflow for targeted next-generation sequencing of low-quality tumor DNA using a 51-gene enrichment panel

Phase Primary objectives Sample set # Samples Sample type DNA input (ng) 1052-Amplicon sequencing platform Confirmation sequencing platform and panel
Workflow Optimization And Platform Evaluation Evaluate analytical performance of the panel Reference DNA mixtures with known genotypes (based on 1000 Genomes Project) 6 (Samples), 6 (Mixtures) Intact 250-2000 GAIIx PGM AmpliSeq
Determine the impact of DNA input quantity
Catalog systematic variants based on Hardy-Weinberg equilibrium. Intact disease free lymphocyte samples 29 Intact 500 GAIIx N/A
Evaluate intact- and FFPE-DNA samples NA12878 and 2 CRC blocks 1 (Intact), 2 (FFPE) Intact, FFPE 250-1000 GAIIx N/A
Demonstrate the impact of input DNA mass on variance
Investigate GC > AT background CRC blocks 8 FFPE 250 GAIIx GAIIx 35-amplicon
Platform Evaluation Using Clinical Specimens Assess performance on clinically relevant FFPE samples Clinical CRC samples 46 FFPE 250 GAIIx PGM 1-amplicon
Assess performance on clinically relevant FFPE samples stratified by QFI Clinical thyroid-cancer samples 72 FFPE 500, 1000 HiSeq Luminex liquid bead array
  1. The first phase of this study was focused on the1052-amplicon panel design, analytical performance testing, and bioinformatics workflow optimization. It was conducted using DNA from intact cell lines with known allele frequencies for analytical and variant detection analysis, and FFPE samples for evaluating platform behavior with low DNA quality samples. The second phase focused on clinical application using FFPE samples from different patient cohorts. Note that amplifiable FFPE samples were quantified using QFI. If an alternative (confirmation) platform was used on a given cohort, both platforms are listed in the last column.