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Validation of a next-generation sequencing oncology panel optimized for low input DNA
- Source :
- Cancer Genetics. :55-63
- Publication Year :
- 2018
- Publisher :
- Elsevier BV, 2018.
-
Abstract
- One caveat of next-generation sequencing (NGS)-based clinical oncology testing is the high amount of input DNA required. We sought to develop a focused NGS panel that could capture hotspot regions in relevant genes requiring 0.5-10 ng input DNA. The resulting Penn Precision Panel (PPP) targeted 20 genes containing clinically significant variants relevant to many cancers. One hundred twenty-three samples were analyzed, including 83 solid tumor specimens derived from FFPE. Various input quantities of DNA (0.5-10 ng) were amplified with content-specific PCR primer pools, then sequenced on a MiSeq instrument (Illumina, Inc.) via paired-end, 2 × 186 base pair reads to an average read depth of greater than 6500x. Variants were detected using an in-house analysis pipeline. Clinical sensitivity and specificity were assessed using results from our previously validated solid tumor NGS panel; sensitivity of the PPP is 96.75% (387/400 variants) and specificity is 99.9% (8427/8428 base pairs). Variant allele frequencies (VAFs) are highly concordant across both assays (r = 0.98 p 0.0001). The PPP is a robust, clinically validated test optimized for low-yield solid tumor specimens, capturing a high percentage of clinically relevant variants found by larger commercially available NGS panels while using only 0.5-10 ng of input DNA.
- Subjects :
- 0301 basic medicine
Clinical Oncology
Cancer Research
Base pair
Low input
High-Throughput Nucleotide Sequencing
Read depth
DNA, Neoplasm
Computational biology
Biology
DNA sequencing
03 medical and health sciences
chemistry.chemical_compound
030104 developmental biology
0302 clinical medicine
chemistry
Limit of Detection
030220 oncology & carcinogenesis
Genetics
Humans
Solid tumor
Molecular Biology
Gene
DNA
Subjects
Details
- ISSN :
- 22107762
- Database :
- OpenAIRE
- Journal :
- Cancer Genetics
- Accession number :
- edsair.doi.dedup.....c94048b96f91d1e2f5b85e9fec9035ad
- Full Text :
- https://doi.org/10.1016/j.cancergen.2018.08.004