Related Experiment Video
Updated: Apr 10, 2026
Integration of Wet and Dry Bench Processes Optimizes Targeted Next-generation Sequencing of Low-quality and Low-quantity Tumor Biopsies
Published on: April 11, 2016
Identification of major factors associated with failed clinical molecular oncology testing performed by next
Hussam Al-Kateb1, TuDung T Nguyen1, Karen Steger-May2
1Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, USA.
Purpose:
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.
Materials And Methods:
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.
Results:
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).
Conclusions:
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.
Insights
Next-generation sequencing (NGS) test failures in cancer patients are often due to insufficient tissue or DNA. Optimizing biopsy acquisition can improve NGS success rates for clinical care.
Area of Science:
- Oncology
- Genomics
- Molecular Diagnostics
Background:
- Next-generation sequencing (NGS) is crucial for guiding cancer patient treatment.
- However, NGS testing is not always successful, and reasons for failure require systematic evaluation.
Purpose of the Study:
- To systematically evaluate factors contributing to NGS test failures in cancer specimens.
- To identify pre-analytical variables impacting the success of comprehensive cancer panels.
Main Methods:
- Analyzed 1528 solid and hematolymphoid tumor specimens tested with an NGS comprehensive cancer panel.
- Extracted DNA and generated 2x101 bp paired-end reads on cancer-related genes using Illumina platforms.
Main Results:
- 22.5% of NGS tests failed, primarily due to insufficient tissue (65%) and insufficient DNA (28.9%).
- Biopsy site, type, clinical setting, specimen age, and DNA quality were significantly associated with test failures.
- Multivariate analysis identified clinical setting and biopsy type as key predictors for insufficient tissue and DNA.
Conclusions:
- Pre-analytical issues like insufficient tissue and DNA cause the majority of NGS test failures.
- Biopsy acquisition factors (clinical setting, site, type, number of cores) significantly influence NGS success.
- Addressing these factors during tissue biopsy collection can enhance NGS analytic success rates.
