BACKGROUND: Massively parallel, or next-generation, sequencing is a powerful technique for the assessment of somatic genomic alterations in cancer samples. Numerous gene targets can be interrogated simultaneously with a high degree of sensitivity. The clinical standard of care for many advanced solid and hematologic malignancies currently requires mutation analysis of several genes in the front-line setting, making focused next generation sequencing (NGS) assays an effective tool for clinical molecular diagnostic laboratories. METHODS: We have utilized an integrated microfluidics circuit (IFC) technology for multiplex PCR-based library preparation coupled with a bioinformatic method designed to enhance indel detection. A parallel low input PCR-based library preparation method was developed for challenging specimens with low DNA yield. Computational data filters were written to optimize analytic sensitivity and specificity for clinically relevant variants. RESULTS: Minimum sequencing coverage and precision of variant calls were the two primary criteria used to establish minimum DNA mass input onto the IFC. Wet-bench and bioinformatics protocols were modified based on data from the optimization and familiarization process to improve assay performance. The NGS platform was then clinically validated for single nucleotide and indel (up to 93 base pair) variant detection with overall analytic accuracy of 98% (97% sensitivity; 100% specificity) using as little as 3 ng of formalin-fixed, paraffin-embedded DNA or 0.3 ng of unfixed DNA. CONCLUSIONS: We created a targeted clinical NGS assay for common solid and hematologic cancers with high sensitivity, high specificity, and the flexibility to test very limited tissue samples often encountered in routine clinical practice.
BACKGROUND: Massively parallel, or next-generation, sequencing is a powerful technique for the assessment of somatic genomic alterations in cancer samples. Numerous gene targets can be interrogated simultaneously with a high degree of sensitivity. The clinical standard of care for many advanced solid and hematologic malignancies currently requires mutation analysis of several genes in the front-line setting, making focused next generation sequencing (NGS) assays an effective tool for clinical molecular diagnostic laboratories. METHODS: We have utilized an integrated microfluidics circuit (IFC) technology for multiplex PCR-based library preparation coupled with a bioinformatic method designed to enhance indel detection. A parallel low input PCR-based library preparation method was developed for challenging specimens with low DNA yield. Computational data filters were written to optimize analytic sensitivity and specificity for clinically relevant variants. RESULTS: Minimum sequencing coverage and precision of variant calls were the two primary criteria used to establish minimum DNA mass input onto the IFC. Wet-bench and bioinformatics protocols were modified based on data from the optimization and familiarization process to improve assay performance. The NGS platform was then clinically validated for single nucleotide and indel (up to 93 base pair) variant detection with overall analytic accuracy of 98% (97% sensitivity; 100% specificity) using as little as 3 ng of formalin-fixed, paraffin-embedded DNA or 0.3 ng of unfixed DNA. CONCLUSIONS: We created a targeted clinical NGS assay for common solid and hematologic cancers with high sensitivity, high specificity, and the flexibility to test very limited tissue samples often encountered in routine clinical practice.
Authors: Garrett M Frampton; Siraj M Ali; Mark Rosenzweig; Juliann Chmielecki; Xinyuan Lu; Todd M Bauer; Mikhail Akimov; Jose A Bufill; Carrie Lee; David Jentz; Rick Hoover; Sai-Hong Ignatius Ou; Ravi Salgia; Tim Brennan; Zachary R Chalmers; Savina Jaeger; Alan Huang; Julia A Elvin; Rachel Erlich; Alex Fichtenholtz; Kyle A Gowen; Joel Greenbowe; Adrienne Johnson; Depinder Khaira; Caitlin McMahon; Eric M Sanford; Steven Roels; Jared White; Joel Greshock; Robert Schlegel; Doron Lipson; Roman Yelensky; Deborah Morosini; Jeffrey S Ross; Eric Collisson; Malte Peters; Philip J Stephens; Vincent A Miller Journal: Cancer Discov Date: 2015-05-13 Impact factor: 39.397
Authors: Lawrence J Jennings; Maria E Arcila; Christopher Corless; Suzanne Kamel-Reid; Ira M Lubin; John Pfeifer; Robyn L Temple-Smolkin; Karl V Voelkerding; Marina N Nikiforova Journal: J Mol Diagn Date: 2017-03-21 Impact factor: 5.568
Authors: Somak Roy; Christopher Coldren; Arivarasan Karunamurthy; Nefize S Kip; Eric W Klee; Stephen E Lincoln; Annette Leon; Mrudula Pullambhatla; Robyn L Temple-Smolkin; Karl V Voelkerding; Chen Wang; Alexis B Carter Journal: J Mol Diagn Date: 2017-11-21 Impact factor: 5.568
Authors: M Nakao; S Yokota; T Iwai; H Kaneko; S Horiike; K Kashima; Y Sonoda; T Fujimoto; S Misawa Journal: Leukemia Date: 1996-12 Impact factor: 11.528
Authors: Simon A Forbes; David Beare; Harry Boutselakis; Sally Bamford; Nidhi Bindal; John Tate; Charlotte G Cole; Sari Ward; Elisabeth Dawson; Laura Ponting; Raymund Stefancsik; Bhavana Harsha; Chai Yin Kok; Mingming Jia; Harry Jubb; Zbyslaw Sondka; Sam Thompson; Tisham De; Peter J Campbell Journal: Nucleic Acids Res Date: 2016-11-28 Impact factor: 16.971
Authors: Emil Lou; Joanne Xiu; Yasmine Baca; Andrew C Nelson; Benjamin A Weinberg; Muhammad Shaalan Beg; Mohamed E Salem; Heinz-Josef Lenz; Philip Philip; Wafik S El-Deiry; W Michael Korn Journal: Cells Date: 2021-05-21 Impact factor: 6.600