A novel approach to improve detection of somatic DNA variants in solid tumors by next-generation sequencing from FFPE samples.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e22177-e22177 ◽  
Author(s):  
Nitin Udar ◽  
Robert Haigis ◽  
Thomas Gros ◽  
Agda Karina Lucio-Eterovic ◽  
Qingxiu Zhang ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Deep Sequencing ◽  
False Positive ◽  
Limit Of Detection ◽  
Low Frequency ◽  
Sample Type ◽  
Next Generation ◽  
Novel Approach ◽  
Ffpe Samples ◽  
Generation Sequencing

e22177 Background: Low frequency variant detection by sequencing is a highly desired goal for therapy selection in cancer especially the detection of actionable targets. The lower limit of detection using Sanger sequening is ~20% minor allele frequency (MAF). Deep sequencing of target genes using next generation sequencing (NGS) is gaining popularity. Formalin Fixed Paraffin Embedded (FFPE) tissue is the most common sample type in solid tumor histopathology. However, because the fixation process fragments DNA and damages it at varying frequencies, downstream processes can potentially misclassify modified bases and generate artifacts. We have developed a protocol that addresses both of these issues in a multiplex assay that involves deep sequencing using NGS of targets implicated in lung, gastric, colon, melanoma and ovarian cancers. Methods: The sample set includes 168 FFPE samples and 82 non FFPE samples. TruSeq Custom Amplicon technology was used to generate libraries for targets across 26 genes. Deep sequencing was done on a NGS platform (Illumina-MiSeq). Results: The DNA quality test, which surveys multiple genomic targets by qPCR, was an accurate determinant of DNA amplifiability and yielded a 99% sample success rate. A sensitivity of <5% MAF was achieved by sequencing at a depth of 1,000X for all targets. In order to differentiate true low frequency variants from fixation and other artifacts, our novel approach investigates each of the two DNA strands independently. The information is bioinformatically combined to distinguish true variants from artifacts. Testing of the FFPE samples with a 5% MAF cut off using the two strand approach reduced the potential false positive rate by ~ 40% when compared to information from only one strand of DNA. A comparative analysis of matched FF and FFPE sample showed that a high percentage of false positive calls were present even in the fresh frozen samples at this high level of sensitivity, if only using information from one strand. Conclusions: This protocol, efficiently and accurately detect low frequency variants by NGS in DNA extracted from FFPE tissues.

scholarly journals Next-Generation Sequencing for Pathogen Identification in Infected Foot Ulcers

2020 ◽  
Vol 5 (4) ◽  
pp. 2473011420S0002
Author(s):  
Yoonjung Choi ◽  
Irvin Oh
Keyword(s):  
Next Generation Sequencing ◽  
False Positive ◽  
Bacterial Genome ◽  
Rrna Gene ◽  
Next Generation ◽  
Antibiotic Resistant ◽  
Streptococcus Species ◽  
Resistant Genes ◽  
Standard Culture ◽  
Generation Sequencing

Category: Other Introduction/Purpose: Foot infections are often polymicrobial with diverse microbiomes. Accurate identification of the main pathogen in diabetic foot ulcer (DFU) remain challenging due to contamination or negative cultures often leading to ineffective post-surgical antibiotic treatment. Application of molecular diagnostics, such as next generation sequencing (NGS) has been explored as an alternative to standard culture in orthopaedic infections. NGS is highly sensitive and detects an entire bacterial genome along with pharmacologic resistant genes in a given sample. We sought to investigate the potential use of NGS for accurate diagnosis and quantification of various species in infected DFU. We hypothesize that NGS will provide a more accurate means of diagnosing and profiling microorganisms in infected DFU compared to the standard culture method. Methods: We investigated 30 infected DFU patients who underwent surgical treatment by a single academic orthopaedic surgeon from October 2018 to September 2019. The average age of the patient was 60.4 (range 33-82) years-old. Surgical procedures performed were irrigation and debridement (12), toe or ray amputation (13), calcanectomies (4), and below-knee amputation (1). Infected bone specimens were obtained intraoperatively and processed for standard culture and NGS. Quantitative PCR was performed to determine the bacterial burden present in the sample. DNA was amplified by PCR from a highly conserved region of the rRNA gene in the bacteria (16S rRNA). Once a high level of DNA was generated and determined, it was compared against NIH GenBank database. Concordance between the standard culture and NGS was assessed. Results: In 28 of 29 patients, pathogens were identified by both NGS and culture, with complete consistency of organisms in 13 cases (concordance rate: 43.3%). NGS provided relative quantitative measures and the presence of antibiotic resistant genes for each pathogen. In NGS, Anaerococcus species (79.3%) was the most common organism, followed by Streptococcus species (44.8%), Prevotella species (44.8%), Finegoldia magna (44.8%). In culture, S. aureus (58.6%) was the most common, followed by Streptococcus species (34.5%), coagulase-negative Staphylococci (24.1%), Corynebacterium species (20.7%). On average, NGS revealed 5.1 (1-11) number of pathogens, whereas standard culture revealed 2.6 (1-6) pathogens in a given sample. NGS identified 2 cases with false positive standard culture and detected antibiotic resistant organisms in 15 specimens. Conclusion: NGS is an emerging method of microbial identification in orthopedic infection. It is particularly helpful in profiling diverse microbes in polymicrobial infected DFU. It can identify major pathogens and may correct false positive or false negative culture. NGS may allow a faster invitation of postoperative targeted antibiotic therapy. [Table: see text]

Does histological assessment accurately distinguish separate primary lung adenocarcinomas from intrapulmonary metastases? A study of paired resected lung nodules in 32 patients using a routine next-generation sequencing panel for driver mutations

2021 ◽  
pp. jclinpath-2021-207421
Author(s):  
Frido K Bruehl ◽  
Erika E Doxtader ◽  
Yu-Wei Cheng ◽  
Daniel H Farkas ◽  
Carol Farver ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Molecular Analysis ◽  
Low Frequency ◽  
Driver Mutations ◽  
Lung Nodules ◽  
Driver Gene ◽  
Next Generation ◽  
Histological Assessment ◽  
Lung Adenocarcinomas ◽  
Generation Sequencing

AimVarious approaches have been reported for distinguishing separate primary lung adenocarcinomas from intrapulmonary metastases in patients with two lung nodules. The aim of this study was to determine whether histological assessment is reliable and accurate in distinguishing separate primary lung adenocarcinomas from intrapulmonary metastases using routine molecular findings as an adjunct.MethodsWe studied resected tumour pairs from 32 patients with lung adenocarcinomas in different lobes. In 15 of 32 tumour pairs, next-generation sequencing (NGS) for common driver mutations was performed on both nodules. The remainder of tumour pairs underwent limited NGS, or EGFR genotyping. Tumour pairs with different drivers (or one driver/one wild-type) were classified as molecularly unrelated, while those with identical low-frequency drivers were classified as related. Three pathologists independently and blinded to the molecular results categorised tumour pairs as related or unrelated based on histological assessment.ResultsOf 32 pairs, 15 were classified as related by histological assessment, and 17 as unrelated. Of 15 classified as related by histology, 6 were classified as related by molecular analysis, 4 were unrelated and 5 were indeterminate. Of 17 classified as unrelated by histology, 14 were classified as unrelated by molecular analysis, none was related and 3 were indeterminate. Histological assessment of relatedness was inaccurate in 4/32 (12.5%) tumour pairs.ConclusionsA small but significant subset of two-nodule adenocarcinoma pairs is inaccurately judged as related by histological assessment, and can be proven to be unrelated by molecular analysis (driver gene mutations), leading to significant downstaging.

scholarly journals Detection of genome-wide low-frequency mutations with Paired-End and Complementary Consensus Sequencing (PECC-Seq) revealed end-repair derived artifacts as residual errors

2019 ◽  
Author(s):  
Xinyue You ◽  
Suresh Thiruppathi ◽  
Weiying Liu ◽  
Yiyi Cao ◽  
Mikihiko Naito ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Low Frequency ◽  
Repair Process ◽  
Sequencing Error ◽  
Base Substitution ◽  
Next Generation ◽  
Background Error ◽  
Technical Errors ◽  
Genome Wide ◽  
Generation Sequencing

ABSTRACTTo improve the accuracy and the cost-efficiency of next-generation sequencing in ultralow-frequency mutation detection, we developed the Paired-End and Complementary Consensus Sequencing (PECC-Seq), a PCR-free duplex consensus sequencing approach. PECC-Seq employed shear points as endogenous barcodes to identify consensus sequences from the overlap in the shortened, complementary DNA strands-derived paired-end reads for sequencing error correction. With the high accuracy of PECC-Seq, we identified the characteristic base substitution errors introduced by the end-repair process of mechanical fragmentation-based library preparations, which were prominent at the terminal 6 bp of the library fragments in the 5’-NpCpA-3’ or 5’-NpCpT-3’ trinucleotide context. As demonstrated at the human genome scale (TK6 cells), after removing these potential end-repair artifacts from the terminal 6 bp, PECC-Seq could reduce the sequencing error frequency to mid-10−7 with a relatively low sequencing depth. For TA base pairs, the background error rate could be suppressed to mid-10−8. In mutagen-treated TK6, slight increases in mutagen treatment-related mutant frequencies could be detected, indicating the potential of PECC-Seq in detecting genome-wide ultra-rare mutations. In addition, our finding on the patterns of end-repair artifacts may provide new insights in further reducing technical errors not only for PECC-Seq, but also for other next-generation sequencing techniques.

scholarly journals Next-Generation Sequencing and Bioinformatics Protocol for Malaria Drug Resistance Marker Surveillance

2018 ◽  
Vol 62 (4) ◽  
pp. e02474-17 ◽  
Author(s):  
Eldin Talundzic ◽  
Shashidhar Ravishankar ◽  
Julia Kelley ◽  
Dhruviben Patel ◽  
Mateusz Plucinski ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Next Generation Sequencing ◽  
Deep Sequencing ◽  
Artemisinin Resistance ◽  
The United States ◽  
Imported Malaria ◽  
Chloroquine Resistance ◽  
Next Generation ◽  
Content Type ◽  
Generation Sequencing

ABSTRACT The recent advances in next-generation sequencing technologies provide a new and effective way of tracking malaria drug-resistant parasites. To take advantage of this technology, an end-to-end Illumina targeted amplicon deep sequencing (TADS) and bioinformatics pipeline for molecular surveillance of drug resistance in P. falciparum, called malaria resistance surveillance (MaRS), was developed. TADS relies on PCR enriching genomic regions, specifically target genes of interest, prior to deep sequencing. MaRS enables researchers to simultaneously collect data on allele frequencies of multiple full-length P. falciparum drug resistance genes (crt, mdr1, k13, dhfr, dhps, and the cytochrome b gene), as well as the mitochondrial genome. Information is captured at the individual patient level for both known and potential new single nucleotide polymorphisms associated with drug resistance. The MaRS pipeline was validated using 245 imported malaria cases that were reported to the Centers for Disease Control and Prevention (CDC). The chloroquine resistance crt CVIET genotype (mutations underlined) was observed in 42% of samples, the highly pyrimethamine-resistant dhps IRN triple mutant in 92% of samples, and the sulfadoxine resistance dhps mutation SGEAA in 26% of samples. The mdr1 NFSND genotype was found in 40% of samples. With the exception of two cases imported from Cambodia, no artemisinin resistance k13 alleles were identified, and 99% of patients carried parasites susceptible to atovaquone-proguanil. Our goal is to implement MaRS at the CDC for routine surveillance of imported malaria cases in the United States and to aid in the adoption of this system at participating state public health laboratories, as well as by global partners.

scholarly journals Differences in Mutational Profile between Follicular Thyroid Carcinoma and Follicular Thyroid Adenoma Identified Using Next Generation Sequencing

2019 ◽  
Vol 20 (13) ◽  
pp. 3126 ◽  
Author(s):  
Martyna Borowczyk ◽  
Ewelina Szczepanek-Parulska ◽  
Szymon Dębicki ◽  
Bartłomiej Budny ◽  
Frederik A. Verburg ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
P Value ◽  
Thyroid Adenoma ◽  
Next Generation ◽  
Mutational Status ◽  
Follicular Thyroid Adenoma ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
Follicular Lesions ◽  
Generation Sequencing

We aimed to identify differences in mutational status between follicular thyroid adenoma (FTA) and follicular thyroid cancer (FTC). The study included 35 patients with FTA and 35 with FTC. DNA was extracted from formalin-fixed paraffin-embedded (FFPE) samples from thyroidectomy. Next-generation sequencing (NGS) was performed with the 50-gene Ion AmpliSeq Cancer Hotspot Panel v2. Potentially pathogenic mutations were found in 14 (40%) FTA and 24 (69%) FTC patients (OR (95%CI) = 3.27 (1.22−8.75)). The number of mutations was higher in patients with FTC than FTA (p-value = 0.03). SMAD4 and STK11 mutations were present only in patients with FTA, while defects in FBXW7, JAK3, KIT, NRAS, PIK3CA, SMARCB1, and TP53 were detected exclusively in FTC patients. TP53 mutations increased the risk of FTC; OR (95%CI) = 29.24 (1.64–522.00); p-value = 0.001. FLT3-positivity was higher in FTC than in the FTA group (51.4% vs. 28.6%; p-value = 0.051). The presence of FLT3 and TP53 with no RET mutations increased FTC detectability by 17.1%, whereas the absence of FLT3 and TP53 with a presence of RET mutations increased FTA detectability by 5.7%. TP53 and FLT3 are candidate markers for detecting malignancy in follicular lesions. The best model to predict FTA and FTC may consist of FLT3, TP53, and RET mutations considered together.

Comparison between two different next generation sequencing platforms for clinical relevant gene mutation test in solid tumours

2020 ◽  
Vol 73 (9) ◽  
pp. 602-604
Author(s):  
Silvia Bessi ◽  
Francesco Pepe ◽  
Marco Ottaviantonio ◽  
Pasquale Pisapia ◽  
Umberto Malapelle ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
High Performance ◽  
Solid Tumours ◽  
Next Generation ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Thermo Fisher Scientific ◽  
Formalin Fixed Paraffin Embedded ◽  
Sequencing Platforms ◽  
Generation Sequencing

In the present study, we analysed 44 formalin fixed paraffin embedded (FFPE) from different solid tumours by adopting two different next generation sequencing platforms: GeneReader (QIAGEN, Hilden, Germany) and Ion Torrent (Thermo Fisher Scientific, Waltham, Massachusetts, USA). We highlighted a 100% concordance between the platforms. In addition, focusing on variant detection, we evaluated a very good agreement between the two tests (Cohen’s kappa=0.84) and, when taking into account variant allele fraction value for each variant, a very high concordance was obtained (Pearson’s r=0.94). Our results underlined the high performance rate of GeneReader on FFPE samples and its suitability in routine molecular predictive practice.

scholarly journals Towards standardization of next-generation sequencing of FFPE samples for clinical oncology: intrinsic obstacles and possible solutions

2017 ◽  
Vol 15 (1) ◽  
Author(s):  
Maxim Ivanov ◽  
Konstantin Laktionov ◽  
Valery Breder ◽  
Polina Chernenko ◽  
Ekaterina Novikova ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Clinical Oncology ◽  
Next Generation ◽  
Ffpe Samples ◽  
Generation Sequencing
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