scholarly journals Sequence Artifacts in DNA from Formalin-Fixed Tissues: Causes and Strategies for Minimization

2015 ◽  
Vol 61 (1) ◽  
pp. 64-71 ◽  
Author(s):  
Hongdo Do ◽  
Alexander Dobrovic
Keyword(s):  
Dna Analysis ◽  
Massively Parallel Sequencing ◽  
Amplicon Sequencing ◽  
Careful Consideration ◽  
Dna Lesions ◽  
Massively Parallel ◽  
Cytosine Deamination ◽  
Accurate Detection ◽  
Ffpe Tissues ◽  
Formalin Fixed

Abstract BACKGROUND Precision medicine is dependent on identifying actionable mutations in tumors. Accurate detection of mutations is often problematic in formalin-fixed paraffin-embedded (FFPE) tissues. DNA extracted from formalin-fixed tissues is fragmented and also contains DNA lesions that are the sources of sequence artifacts. Sequence artifacts can be difficult to distinguish from true mutations, especially in the context of tumor heterogeneity, and are an increasing interpretive problem in this era of massively parallel sequencing. Understanding of the sources of sequence artifacts in FFPE tissues and implementation of preventative strategies are critical to improve the accurate detection of actionable mutations. CONTENT This mini-review focuses on DNA template lesions in FFPE tissues as the source of sequence artifacts in molecular analysis. In particular, fragmentation, base modification (including uracil and thymine deriving from cytosine deamination), and abasic sites are discussed as indirect or direct sources of sequence artifacts. We discuss strategies that can be implemented to minimize sequence artifacts and to distinguish true mutations from sequence artifacts. These strategies are applicable for the detection of actionable mutations in both single amplicon and massively parallel amplicon sequencing approaches. SUMMARY Because FFPE tissues are usually the only available material for DNA analysis, it is important to maximize the accurate informational content from FFPE DNA. Careful consideration of each step in the work flow is needed to minimize sequence artifacts. In addition, validation of actionable mutations either by appropriate experimental design or by orthogonal methods should be considered.

scholarly journals Reducing Sequence Artifacts in Amplicon-Based Massively Parallel Sequencing of Formalin-Fixed Paraffin-Embedded DNA by Enzymatic Depletion of Uracil-Containing Templates

2013 ◽  
Vol 59 (9) ◽  
pp. 1376-1383 ◽  
Author(s):  
Hongdo Do ◽  
Stephen Q Wong ◽  
Jason Li ◽  
Alexander Dobrovic
Keyword(s):  
Massively Parallel Sequencing ◽  
Significant Proportion ◽  
Massively Parallel ◽  
Sequencing Error ◽  
Parallel Sequencing ◽  
Cpg Dinucleotides ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Tissues ◽  
Formalin Fixed

BACKGROUND Formalin-fixed, paraffin-embedded (FFPE) tissues are routinely used for detecting mutational biomarkers in patients with cancer. A previous intractable challenge with FFPE DNA in genetic testing has been the high number of artifactual single-nucleotide changes (SNCs), particularly for the detection of low-level mutations. Pretreatment of FFPE DNA with uracil-DNA glycosylase (UDG) can markedly reduce these C:G>T:A SNCs with a small panel of amplicons. This procedure has implications for massively parallel sequencing approaches to mutation detection from DNA. We investigated whether sequence artifacts were problematic in amplicon-based massively parallel sequencing and what effect UDG pretreatment had on reducing these artifacts. METHODS We amplified selected amplicons from lung cancer FFPE DNAs using the TruSeq Cancer Panel. SNCs occurring at a frequency <10% were considered most likely to represent sequence artifacts and were enumerated for both UDG-treated and -untreated DNAs. RESULTS Massively parallel sequencing of FFPE DNA samples showed multiple SNCs, predominantly C:G>T:A changes, with a significant proportion occurring above the BACKGROUND sequencing error (defined as 1%). UDG pretreatment markedly reduced C:G>T:A SNCs without affecting the detection of true somatic mutations. However, C:G>T:A changes within CpG dinucleotides were often resistant to the UDG treatment as a consequence of 5-methyl cytosine being deaminated to thymine rather than uracil. CONCLUSIONS UDG pretreatment greatly facilitates the accurate discrimination of mutations in FFPE samples by use of amplicon-based approaches. This is particularly important when working with samples with low tumor purity or for the assessment of mutational heterogeneity in tumors.

Identifying cancer mutations in neuroendocrine prostate cancer (NEPC) through massively parallel DNA sequencing of formalin-fixed paraffin-embedded (FFPE) tissue.

2012 ◽  
Vol 30 (5_suppl) ◽  
pp. 110-110
Author(s):  
Himisha Beltran ◽  
Kyung Park ◽  
Scott T. Tagawa ◽  
Roman Yelensky ◽  
Doron Lipson ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Dna Sequencing ◽  
Dna Analysis ◽  
Massively Parallel Sequencing ◽  
Ffpe Tissue ◽  
Massively Parallel ◽  
Sequence Coverage ◽  
High Confidence ◽  
Biopsy Material ◽  
Synonymous Mutations

110 Background: NEPC is an aggressive and lethal variant of prostate cancer that most commonly arises from existing prostate adenocarcinoma (PCA). Little is known about the underlying biology of NEPC, as metastases are rarely biopsied. The purpose of this study is to determine the spectrum of somatic mutations in NEPC by using a novel DNA sequencing platform and to evaluate for targetable molecular alterations. Methods: 44 NEPC and mixed NEPC/PCA were evaluated and 77 high density foci of NEPC, PCA, and benign areas were selected for DNA extraction. Massively parallel sequencing via HiSeq2000 was performed using indexed seq libraries constructed by adapter ligation followed by hybridization with optimized capture probes. Data was processed using publicly available and newly validated software tools to make mutation assignments for base alterations, indels, and CNAs in 182 cancer associated genes and common sites of rearrangement for 15 genes. Results: 5/8 biopsies and 62/65 (overall 93%) of prostate foci yielded sufficient DNA (>50 ng) for analysis from 40m of FFPE tissue. Average median sequence coverage was 934x. TMPRSS2-ERG fusion was present in 28% of NEPC. Recurrent homozygous deletions involving PTEN and RB1 were seen, and 1 tumor with BRCA2 loss. Several high confidence non-synonymous mutations were identified including TP53 (40%), CTNNB1 (12%), and less frequently mutations involving PTEN, PIK3CA, AR, as well as other novel mutations/fusions. There was high concordance between NEPC and PCA foci in mixed tumors, as well as between primary tumor and metastases. High confidence lesions were validated with exome sequencing and FISH. Conclusions: This study shows feasibility of an in-depth DNA analysis using FFPE tissue, and even biopsy material. Comprehensive genome sequencing has nominated novel biologic pathways and provides insight into disease progression from PCA to NEPC, as well as potential new drug targets for a tumor that is currently lethal. This is a useful and comprehensive sequencing tool to evaluate tumors such as NEPC and other metastatic tumors, where obtaining tissue is challenging.

Abstract 3938: Massively parallel sequencing of formalin-fixed, paraffin-embedded tissue

2011 ◽  
Author(s):  
Richard B. Everson ◽  
Heather R. Glatt-Deeley ◽  
Linda C. Burian ◽  
Lorrie A. Perpetua ◽  
Upendra P. Hegde ◽  
...  
Keyword(s):  
Massively Parallel Sequencing ◽  
Massively Parallel ◽  
Parallel Sequencing ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

scholarly journals Laboratory Verification of a BRCA1 and BRCA2 Massively Parallel Sequencing Assay from Wet Bench to Bioinformatics for Germline DNA Analysis

2021 ◽  
Author(s):  
Kok-Siong Poon ◽  
Lily Chiu ◽  
Karen Mei-Ling Tan
Keyword(s):  
Genetic Testing ◽  
Sanger Sequencing ◽  
Dna Analysis ◽  
Massively Parallel Sequencing ◽  
Massively Parallel ◽  
Brca1 And Brca2 ◽  
Clinical Diagnostic Laboratory ◽  
Parallel Sequencing ◽  
Miseq Platform ◽  
Brca Genetic Testing

Abstract Introduction A robust genetic test for BRCA1 and BRCA2 genes is necessary for the diagnosis, prognosis, and treatment of patients with hereditary breast and ovarian cancer. We evaluated a commercial amplicon-based massively parallel sequencing (MPS) assay, BRCA MASTR Plus on the MiSeq platform, for germline BRCA genetic testing. Methods This study was performed on 31 DNA from cell lines and proficiency testing samples to establish the accuracy of the assay. A reference cell line DNA, NA12878 was used to determine the reproducibility of the assay. Discordant MPS result was resolved orthogonally by the current gold-standard Sanger sequencing method. Results The analytical accuracy, sensitivity, and specificity for variant detection were 93.55, 92.86, and 100.00%, respectively. Both sequencing depth and variant allele frequencies were highly reproducible by comparing the NA12878 DNA tested in three separate runs. The single discordant result, later confirmed by Sanger sequencing was due to the inability of the MASTR Reporter software to identify a 40-bp deletion in BRCA1. Conclusion The BRCA MASTR Plus assay on the MiSeq platform is accurate and reproducible for germline BRCA genetic testing, making it suitable for use in a clinical diagnostic laboratory. However, Sanger sequencing may still serve as a confirmatory method to improve diagnostic capability of the MPS assay.

scholarly journals High-Resolution Profiling of Fetal DNA Clearance from Maternal Plasma by Massively Parallel Sequencing

2013 ◽  
Vol 59 (8) ◽  
pp. 1228-1237 ◽  
Author(s):  
Stephanie CY Yu ◽  
Shara WY Lee ◽  
Peiyong Jiang ◽  
Tak Y Leung ◽  
KC Allen Chan ◽  
...  
Keyword(s):  
High Resolution ◽  
Half Life ◽  
Dna Analysis ◽  
Massively Parallel Sequencing ◽  
Maternal Plasma ◽  
Massively Parallel ◽  
Slow Phase ◽  
Parallel Sequencing ◽  
Fetal Dna ◽  
Circulating Fetal Dna

BACKGROUND With the advent of massively parallel sequencing (MPS), DNA analysis can now be performed in a genomewide manner. Recent studies have demonstrated the high precision of MPS for quantifying fetal DNA in maternal plasma. In addition, paired-end sequencing can be used to determine the size of each sequenced DNA fragment. We applied MPS in a high-resolution investigation of the clearance profile of circulating fetal DNA. METHODS Using paired-end MPS, we analyzed serial samples of maternal plasma collected from 13 women after cesarean delivery. We also studied the transrenal excretion of circulating fetal DNA in 3 of these individuals by analyzing serial urine samples collected after delivery. RESULTS The clearance of circulating fetal DNA occurred in 2 phases, with different kinetics. The initial rapid phase had a mean half-life of approximately 1 h, whereas the subsequent slow phase had a mean half-life of approximately 13 h. The final disappearance of circulating fetal DNA occurred at about 1 to 2 days postpartum. Although transrenal excretion was involved in the clearance of circulating fetal DNA, it was not the major route. Furthermore, we observed significant changes in the size profiles of circulating maternal DNA after delivery, but we did not observe such changes in circulating fetal DNA. CONCLUSIONS MPS of maternal plasma and urinary DNA permits high-resolution study of the clearance profile of circulating fetal DNA.

Harnessing the potential of the environmental microbiome in forensic science

2021 ◽  
pp. 61-67
Author(s):  
Agata Jagiełło ◽  
◽  
Anna Woźniak ◽  
Błażej Szczerba ◽  
Rafał Płoski ◽  
...  
Keyword(s):  
Forensic Science ◽  
Dna Analysis ◽  
Genetic Test ◽  
Massively Parallel Sequencing ◽  
Geographical Location ◽  
Soil Samples ◽  
Massively Parallel ◽  
Soil Microbiome ◽  
Microbiome Analysis ◽  
Dna Profiles

A scientific consortium led by the Central Forensic Laboratory of the Police has undertaken to develop a method for DNA analysis of the soil microbiome to be used in forensic investigations. The aim of the project entitled Soil Microbiome Analysis Forensic Tool – SMAFT (http://smaft.eu/), financed by the National Center for Research and Development (DOB-BIO10/03/01/2019), is to develop a new tool that enables the association of a trace in the form of a soil sample with a specific geographical location. The first part of the paper introduces the concept of the microbiome and presents the possibilities of using microbiome DNA analysis in forensic science. In the second part, the stages of the SMAFT project are described in detail, beginning from the collection of soil samples from different sites in Poland across all seasons and isolation of microbiome DNA through massively parallel sequencing (MPS) technology-based analysis of isolates and the development of a genetic test containing a set of metagenomic markers allowing for effective individualization of soil samples, up to the creation of an IT system enabling analysis and interpretation of the obtained results, which includes a database of soil microbiome DNA profiles from various locations in Poland.

scholarly journals Massively parallel sequencing of formalin-fixed paraffin-embedded tissues

2012 ◽  
Vol 6 (S6) ◽  
Author(s):  
Rachel L Erlich ◽  
Emanuele Palescandolo ◽  
Robert T Jones ◽  
Ashwini Sunkavalli ◽  
Alina Raza ◽  
...  
Keyword(s):  
Massively Parallel Sequencing ◽  
Massively Parallel ◽  
Parallel Sequencing ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed
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