Validation of Next Generation Sequencing Cancer Panels for Clinical Somatic Mutation Profiling-- Identification of Source of Variations and Artifacts using FFPE Tissues

Chang, Ken C. N.; Zhao, Yun

doi:10.4172/2469-9853.1000109

Cited by 5 publications

(23 citation statements)

References 23 publications

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“…Our un-published studies are in agreement with this finding as mutation calls from three library preparations from the same batch of FFPE tissue DNA showed less than 50% of all variant calls generated from WES were shared by the three replicates even though high quality filters were applied (Chang et al manuscript in preparation). Our results also indicate that using independent library preparation replicates is an effective way to identify false positive calls [5]. Recently, O'Rawe et al showed that NGS analysis of the same data set using different variant caller pipelines often resulted in low concordance [7].…”

mentioning

confidence: 69%

“…These false positive calls are not due to result from a lack of repeatability in the sequencing since most current widely-accepted NGS technologies permit excellent instrument run-torun repeatability if same library preparation is used [5]. Instead, the genesis of the false positive calls lies in the library preparation step.…”

Section: Short Communicationmentioning

confidence: 99%

“…Instead, the genesis of the false positive calls lies in the library preparation step. For example, when different library preparations are involved, even starting with same DNA or RNA stock solution, the reproducibility of potential somatic mutation calls (whose variant frequencies usually fall below 25%) is in general poor [5]. For WES this seems particularly concerning, considering the work of Belkadi et al who showed that more than 50% of high quality variant calls were false positives by using Sanger sequencing to confirm randomly selected single nucleotide variant calls [6].…”

Section: Short Communicationmentioning

confidence: 99%

“…Our recent publication revealed the highly fragmented nature of DNA in FFPE tissue samples and the subsequent amplification of the short fragments during library preparation are responsible for many false positive variant calls [5]. These false positive calls are not due to result from a lack of repeatability in the sequencing since most current widely-accepted NGS technologies permit excellent instrument run-torun repeatability if same library preparation is used [5].…”

Section: Short Communicationmentioning

confidence: 99%

“…While we found hotspot mutations were called consistently, the same was not true for non-hotspot locations since many non-hotspot variant calls generated from individual testing FFPE were not reproducible. In addition, among the reproducible variant calls some were platform or panel-specific artifacts that showed up across all samples including reference samples (Chang et al manuscript to be submitted).Our recent publication revealed the highly fragmented nature of DNA in FFPE tissue samples and the subsequent amplification of the short fragments during library preparation are responsible for many false positive variant calls [5]. These false positive calls are not due to result from a lack of repeatability in the sequencing since most current widely-accepted NGS technologies permit excellent instrument run-torun repeatability if same library preparation is used [5].…”

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confidence: 99%

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Next Generation Sequencing and Its Clinical Applications: The Growing Pains

Chang¹,

Marton²

2016

Next Generat Sequenc & Applic

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Short CommunicationAs the list of applications of next generation sequencing (NGS)-based assays continues to grow and the user network continues to expand from academic and pharmaceutical discovery research to clinical decision-making tests, the challenges and controversies continue to persist. Over the past year, the FDA held several workshops on the analytical and clinical validation of NGS tests that resulted in the release of two guidance documents, for which it has requested feedback from NGS stakeholders [1,2]. One key area of debate is defining the best method for establishing analytical validity of NGS tests (use of standards or the use of processes such as quality system regulations, QSRs). Some feel that QSRs are overly burdensome, impractical and cost-prohibitive; others feel the use of standards may be insufficiently rigorous. Our concern is how the standards would be implemented. Another question is related to whether or how clinical labs should confirm novel variant calls. For example, if a whole exome sequencing (WES) of a sample results in 500 somatic mutation calls, it would be essentially impossible (both cost prohibitive and time-consuming) to confirm all 500 variants using an orthogonal method. In some clinical or hospital settings, verifying specific decision-making variant calls for a given patient with special disease condition using orthogonal methods might be feasible or justifiable. However, if the assay is to be used for the determination of patients' hyper-mutation status within a clinical trial, it might be unrealistic to confirm every potential variant call.A third controversy is whether clinical databases could be used for clinical validation of novel mutations not in the literature, especially those mutations for which analytical confirmation was not explicitly performed. Unfortunately, if a public database of genotype-phenotype associations were created using historic data with limited variant confirmation or reproducibility measures in place, even if the data were from reputable labs, using such a database to support clinical validity of an NGS-based in vitro diagnostic might have unintended consequences, and could even increase the risk of getting incorrect diagnoses in the clinic [3,4]. Of course, if the database only contained those specific variants directly supported by the clinical evidence of genotype-phenotype association on a variant-by-variant basis, then that might be acceptable and useful. We can illustrate our concern with an extreme example: suppose a patient's sample that has 500 variants derived from its WES data is confirmed to be a responder for a given treatment, are we saying that now all 500 variants are considered validated and should be deposited into this public database? We would argue against this for several reasons, not the least of which would be the lack of confirmation of each mutation.As for the use of standards in NGS-based in vitro diagnostics for germline diseases, although it should have substantial value in terms of serving as control samples...

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confidence: 69%

Section: Short Communicationmentioning

confidence: 99%

Section: Short Communicationmentioning

confidence: 99%

Section: Short Communicationmentioning

confidence: 99%

mentioning

confidence: 99%

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Next Generation Sequencing and Its Clinical Applications: The Growing Pains

Chang¹,

Marton²

2016

Next Generat Sequenc & Applic

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Deamination Effects in Formalin-Fixed, Paraffin-Embedded Tissue Samples in the Era of Precision Medicine

Kim

Park

et al. 2017

The Journal of Molecular Diagnostics

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Deamination of nucleotides causes C:G>T:A changes in formalin-fixed, paraffin-embedded (FFPE) tissue samples and produces false positives during next-generation sequencing (NGS). Uracil DNA glycosylase (UDG) helps eliminate this issue, but the effect of UDG in different tissue preparation conditions has not been rigorously studied. To investigate whether UDG can reduce false-positive single-nucleotide variant (SNV) calls, we used tumor and normal tissues from gastric adenocarcinoma patients prepared using different fixation times and pH conditions. FFPE tumor blocks >10 years were also evaluated for the comparison. We performed semiconductor-based NGS to evaluate nucleotide changes and used UDG to test deamination-related effects. Sequencing quality parameters mildly worsened with prolonged fixation time, acidic pH, and delayed fixation. SNV calls and C:G>T:A changes increased after >48 hours of fixation. In both recently prepared and old FFPE tissue blocks, UDG treatment reduced deamination-induced nucleotide changes. In the recently prepared samples, both high-quality SNVs and mean target coverage were remarkably increased on treatment with UDG. However, the quality of NGS results from old-age samples varied irrespective of UDG treatment. In conclusion, based on our findings, we believe that when performing NGS on recently embedded blocks, it is important to consider that certain poorly fixed samples may be at the risk of being deaminated, which can be corrected with UDG treatment.

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Past, current and future approaches to querying MAPK pathway activation: status and clinical implications

Chang¹,

Marton²

2014

Personalized Medicine

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MAPK pathway activation related to cancer development has drawn a great deal of attention in the field of personalized medicine in recent years. Many different approaches and assays have been developed to query the activation of this pathway and to develop life-saving treatments. The goal of this review article is threefold. First, to provide a brief overview of the many mutation assays that have been used to detect MAPK pathway activation, and to compare pros and cons of these assay platforms. Second, to focus on one custom-designed multiplexing mutation assay that is currently used to support an ongoing clinical trial and to show the novel features of this assay and its relevance in addressing unmet clinical needs. Third, to provide future perspectives of these MAPK pathway gene mutation detection efforts and to suggest how what we have learned from past and current approaches should guide future developments.

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Validation of Next Generation Sequencing Cancer Panels for Clinical Somatic Mutation Profiling-- Identification of Source of Variations and Artifacts using FFPE Tissues

Cited by 5 publications

References 23 publications

Next Generation Sequencing and Its Clinical Applications: The Growing Pains

Next Generation Sequencing and Its Clinical Applications: The Growing Pains

Deamination Effects in Formalin-Fixed, Paraffin-Embedded Tissue Samples in the Era of Precision Medicine

Past, current and future approaches to querying MAPK pathway activation: status and clinical implications

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