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Mol Oncol. 2015 Nov;9(9):1737-43. doi: 10.1016/j.molonc.2015.05.004. Epub 2015 May 29.

Identification of major factors associated with failed clinical molecular oncology testing performed by next generation sequencing (NGS).

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  • 1Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, USA. Electronic address:
  • 2Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, USA.
  • 3Division of Biostatistics, Washington University School of Medicine, St. Louis, USA.



DNA analysis by NGS has become important to direct the clinical care of cancer patients. However, NGS is not successful in all cases, and the factors responsible for test failures have not been systematically evaluated.


A series of 1528 solid and hematolymphoid tumor specimens was tested by an NGS comprehensive cancer panel during 2012-2014. DNA was extracted and 2×101 bp paired-end sequence reads were generated on cancer-related genes utilizing Illumina HiSeq and MiSeq platforms.


Testing was unsuccessful in 343 (22.5%) specimens. The failure was due to insufficient tissue (INST) in 223/343 (65%) cases, insufficient DNA (INS-DNA) in 99/343 (28.9%) cases, and failed library (FL) in 21/343 (6.1%) cases. 87/99 (88%) of the INS-DNA cases had below 10 ng DNA available for testing. Factors associated with INST and INS-DNA failures were site of biopsy (SOB) and type of biopsy (TOB) (both p < 0.0001), and clinical setting of biopsy (CSB, initial diagnosis or recurrence) (p < 0.0001). Factors common to INST and FL were age of specimen (p ≤ 0.006) and tumor viability (p ≤ 0.05). Factors common to INS-DNA and FL were DNA purity and DNA degradation (all p ≤ 0.005). In multivariate analysis, common predictors for INST and INS-DNA included CSB (p = 0.048 and p < 0.0001) and TOB (both p ≤ 0.003), respectively. SOB (p = 0.004) and number of cores (p = 0.001) were specific for INS-DNA, whereas TOB and DNA degradation were associated with FL (p = 0.04 and 0.02, respectively).


Pre-analytical causes (INST and INS-DNA) accounted for about 90% of all failed cases; independent of test design. Clinical setting; site and type of biopsy; and number of cores used for testing all correlated with failure. Accounting for these factors at the time of tissue biopsy acquisition could improve the analytic success rate.


DNA degradation and purity; Diagnostic biopsy; Failed analysis; Molecular oncology testing by NGS; Number of cores; Site and type of biopsy

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