Evaluation of an integrated clinical workflow for targeted next-generation sequencing of low-quality tumor DNA using a 51-gene enrichment panel.
BACKGROUND
Improvements in both performance and cost for next-generation sequencing (NGS) have spurred its rapid adoption for clinical applications. We designed and optimized a pan-cancer target-enrichment panel for 51 well-established oncogenes and tumor suppressors, in conjunction with a bioinformatic pipeline informed by in-process controls and pre- and post-analytical quality control measures.


METHODS
The evaluation of this workflow consisted of sequencing mixtures of intact DNA to establish analytical sensitivity and precision, utilization of heuristics to identify systematic artifacts, titration studies of intact and FFPE samples for input optimization, and incorporation of orthogonal sequencing strategies to increase both positive predictive value and variant detection. We also used 128 FFPE samples to assess clinical accuracy and incorporated the previously described quantitative functional index (QFI) for sample qualification as part of detailing complete system performance.


RESULTS
We observed a concordance correlation coefficient of 0.99 between the observed versus expected percent variant at 250 ng input across 4 independent sequencing runs. A subset of the systematic variants were confirmed to be barely detectable on an independent sequencing platform (Wilcox signed-rank test p-value <10(-16)), and the incorporation of orthogonal sequencing strategies increased the harmonic mean of sensitivity and positive predictive value of mutation detection by 41%. In one cohort of FFPE tumor samples, coverage and inter-platform concordance were positively correlated with the QFI, emphasizing the need for pre-analytical sample quality control to reduce the risk of false positives and negatives. In a separate cohort of FFPE samples, the 51-gene panel achieved 78% sensitivity (95% CI = 56.3, 92.5) with 100% PPV (95% CI = 81.5, 100.0) based on known mutations at 7.9% median abundance. By sequencing specimens using an orthogonal NGS technology, sensitivity was improved to 87.0% (95% CI = 66.4,97.2) while maintaining PPV.


CONCLUSIONS
The results highlight the value of process integration in a comprehensive targeted NGS system, enabling both discovery and diagnostic applications, particularly when sequencing low-quality cancer specimens.
Ashish
A
Choudhary
Elizabeth
E
Mambo
Tiffany
T
Sanford
Michael
M
Boedigheimer
Brian
B
Twomey
Joseph
J
Califano
Andrew
A
Hadd
Kelly S
KS
Oliner
Sylvie
S
Beaudenon
Gary J
GJ
Latham
Alex T
AT
Adai
D002294:Carcinoma, Squamous Cell / Q000235:genetics; D004273:DNA, Neoplasm / Q000235:genetics*; D052138:Genes, Neoplasm; D006258:Head and Neck Neoplasms / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods / Q000592:standards*; D006801:Humans; D016612:Paraffin Embedding; D011786:Quality Control; D017422:Sequence Analysis, DNA / Q000379:methods / Q000592:standards*; D057188:Workflow
D004273:DNA, Neoplasm