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

Table 2 Overview of study objectives and strategy

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		6 (Samples), 6 (Mixtures)	Intact	250-2000	GAIIx	PGM AmpliSeq
	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	NA12878 and 2 CRC blocks	1 (Intact), 2 (FFPE)	Intact, FFPE	250-1000	GAIIx	N/A
	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
Platform Evaluation Using Clinical Specimens	Assess performance on clinically relevant FFPE samples stratified by QFI	Clinical thyroid-cancer samples	72	FFPE	500, 1000	HiSeq	Luminex liquid bead array

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.

ISSN: 1755-8794