scholarly journals Detection of EGFR deletion using unique RepSeq technology

2019 ◽  
Author(s):  
Thuraiayah Vinayagamoorthy ◽  
Dahui Qin ◽  
Fei Ye ◽  
Minghao Zhong
Keyword(s):  
Internal Control ◽  
Sanger Sequencing ◽  
Limit Of Detection ◽  
High Sensitivity ◽  
Wild Type ◽  
Sequencing Technology ◽  
Sequencing Method ◽  
Exon 19 Deletion ◽  
Sensitivity Specificity ◽  
Generation Sequencing

AbstractWe are reporting a novel sequencing technology, RepSeq (Repetitive Sequence), that has high sensitivity, specificity and quick turn-around time. This new sequencing technology is developed by modifying traditional Sanger sequencing technology in several aspects. The first, a homopolymer tail is added to the PCR primer(s), which makes interpreting electropherograms a lot easier than that in traditional Sanger sequencing. The second, an indicator nucleotide is added at the 5’end of the homopolymer tail. In the presence of a deletion, the position of the indicator nucleotide in relation to the wild type confirms the deletion. At the same time, the indicator of the wild type serves as the internal control. Furthermore, the specific design of the PCR and/or sequencing primers will specifically enrich/select mutant alleles, which increases sensitivity and specificity significantly. Based on serial dilution studies, the analytical lower limit of detection was 1.47 copies. A total of 89 samples were tested for EGFR exon 19 deletion, of which 21 were normal blood samples and 68 were samples previously tested by either pyrosequencing or TruSeq Next Generation Sequencing Cancer Panel. There was 100 % concordance among all the samples tested. RepSeq technology has overcome the shortcomings of Sanger sequencing and offers an easy-to-use novel sequencing method for personalized precision medicine.

A novel mutant-allele capture method for enrichment of low abundant cancer mutants.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e14632-e14632
Author(s):  
Rui Lin ◽  
Yue Pu ◽  
Li Mao
Keyword(s):  
Sanger Sequencing ◽  
Deletion Mutant ◽  
Limit Of Detection ◽  
High Sensitivity ◽  
Braf V600e ◽  
Locked Nucleic Acid ◽  
Wild Type ◽  
Cancer Driver ◽  
Exon 19 Deletion ◽  
Exon 19

e14632 Background: High sensitivity detection of cancer mutants can facilitate cancer diagnosis, treatment selection, and disease monitoring. Improved methods to detect low abundance of cancer driver mutants are needed. Methods: We developed a novel method, termed PEAC, with a super high sensitivity to detect low abundant mutant from cancer driver genes through mutant capturing followed by Sanger sequencing. Circulating free DNA (cfDNA) standards containing 20 ng of 5%, 1%, 0% (wild-type), or 40 ng of 0.1% cancer driver mutants were used to establish the method. After pre-amplification of both wild-type and mutant alleles, mutant fragments were specifically bound by biotin-labelled, locked–nucleic acid modified probes, enriched by streptavidin beads, amplified through PCR and sequenced using Sanger for detection. Results: The wild-type standard did not show any mutation signal either with or without PEAC, nor did the standards with 5%, 1%, or 0.1% mutants prior to PEAC enrichment. However, after PEAC enrichment, 5%, 1%, and 0.1% of cancer driver mutants ( EGFR L858R, EGFR T790M, and BRAF V600E) predicting sensitivities to target therapies were enriched to ~90%, ~80% and 30-40% abundance, respectively. In addition to the application for point mutations, PEAC methodology was also used to enrich deletion mutant; 5%, 1%, and 0.1% of EGFR exon 19 deletion, E746_A750del, were all enriched to 90-100% frequency after PEAC. To address the limit of detection of PEAC technology, 100 ng of cfDNA standard containing 0.01% EGFR L858R and EGFR exon 19 deletion mutant were enriched to 15% and 50%, respectively, after PEAC, which indicates up to 5000-fold enrichment for EGFR exon 19 deletion by PEAC technology. Conclusions: PEAC technology can enrich very low abundance of key cancer driver mutants predicting sensitivities to tyrosine kinase inhibitors. PEAC enrichment in combination with Sanger sequencing may guide the application of cancer target therapy and represent a useful technology in the era of precision medicine.

scholarly journals 40 minutes RT-qPCR Assay for Screening Spike N501Y and HV69-70del Mutations

2021 ◽  
Author(s):  
Gulay Korukluoglu ◽  
Mustafa Kolukirik ◽  
Fatma Bayrakdar ◽  
Gozde Girgin Ozgumus ◽  
Ayse Basak Altas ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Internal Control ◽  
Sanger Sequencing ◽  
Limit Of Detection ◽  
Qpcr Assay ◽  
Rapid Screening ◽  
Clinical Samples ◽  
One Step ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

ABSTRACTA one-step reverse transcription and real-time PCR (RT-qPCR) test was developed for rapid screening (40 minutes) of the Spike N501Y and HV69-70del mutations in SARS-CoV-2 positive samples. The test also targets a conserved region of SARS-CoV-2 Orf1ab as an internal control. The samples containing both the N501Y and HV69-70del mutations are concluded as VOC-202012/01 positive. Samples suspected to be positive for B.1.351 or P.1 are the N501Y positive and HV69-70del negative cases. Limit of detection (LOD) of the kit for Orf1ab target is 500 copies/mL, while that of the N501, Y501 and HV69-70del targets are 5000 copies/mL. The developed assay was applied to 165 clinical samples containing SARS-CoV-2 from 32 different lineages. The SARS-CoV-2 lineages were determined via the next-generation sequencing (NGS). The RT-qPCR results were in 100% agreement with the NGS results that 19 samples were N501Y and HV69-70del positive, 10 samples were N501Y positive and HV69-70del negative, 1 sample was N501Y negative and HV69-70del positive, and 135 samples were N501Y and HV69-70del negative. All the VOC-202012/01 positive samples were detected in people who have traveled from England to Turkey. The RT-qPCR test and the Sanger sequencing was further applied to 1000 SARS-CoV-2 positive clinical samples collected in Jan2021 from the 81 different provinces of Turkey. The RT-qPCR results were in 100% agreement with the Sanger sequencing results that 32 samples were N501Y positive and HV69-70del negative, 4 samples were N501Y negative and HV69-70del positive, 964 samples were N501Y and HV69-70del negative. The specificity of the 40 minutes RT-qPCR assay relative to the sequencing-based technologies is 100%. The developed assay is an advantageous tool for timely and representative estimation of the N501Y positive variants’ prevalence because it allows testing a much higher portion of the SARS-CoV-2 positives in much lower time compared to the sequencing-based technologies.

scholarly journals Confirming putative variants at ≤ 5% allele frequency using allele enrichment and Sanger sequencing

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yan Helen Yan ◽  
Sherry X. Chen ◽  
Lauren Y. Cheng ◽  
Alyssa Y. Rodriguez ◽  
Rui Tang ◽  
...  
Keyword(s):  
Sanger Sequencing ◽  
Limit Of Detection ◽  
High Sensitivity ◽  
Sequencing Errors ◽  
Whole Exome ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Fresh Frozen ◽  
The Cost ◽  
Allelic Fraction

AbstractWhole exome sequencing (WES) is used to identify mutations in a patient’s tumor DNA that are predictive of tumor behavior, including the likelihood of response or resistance to cancer therapy. WES has a mutation limit of detection (LoD) at variant allele frequencies (VAF) of 5%. Putative mutations called at ≤ 5% VAF are frequently due to sequencing errors, therefore reporting these subclonal mutations incurs risk of significant false positives. Here we performed ~ 1000 × WES on fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissue biopsy samples from a non-small cell lung cancer patient, and identified 226 putative mutations at between 0.5 and 5% VAF. Each variant was then tested using NuProbe NGSure, to confirm the original WES calls. NGSure utilizes Blocker Displacement Amplification to first enrich the allelic fraction of the mutation and then uses Sanger sequencing to determine mutation identity. Results showed that 52% of the 226 (117) putative variants were disconfirmed, among which 2% (5) putative variants were found to be misidentified in WES. In the 66 cancer-related variants, the disconfirmed rate was 82% (54/66). This data demonstrates Blocker Displacement Amplification allelic enrichment coupled with Sanger sequencing can be used to confirm putative mutations ≤ 5% VAF. By implementing this method, next-generation sequencing can reliably report low-level variants at a high sensitivity, without the cost of high sequencing depth.

scholarly journals High sensitivity sanger sequencing detection of BRAF mutations in metastatic melanoma FFPE tissue specimens

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Lauren Y. Cheng ◽  
Lauren E. Haydu ◽  
Ping Song ◽  
Jianyi Nie ◽  
Michael T. Tetzlaff ◽  
...  
Keyword(s):  
Sanger Sequencing ◽  
Limit Of Detection ◽  
False Negative ◽  
High Sensitivity ◽  
Ffpe Tissue ◽  
Braf Mutations ◽  
Tissue Samples ◽  
Ffpe Samples ◽  
Droplet Pcr ◽  
Tissue Specimens

AbstractMutations in the BRAF gene at or near the p. V600 locus are informative for therapy selection, but current methods for analyzing FFPE tissue DNA generally have a limit of detection of 5% variant allele frequency (VAF), or are limited to the single variant (V600E). These can result in false negatives for samples with low VAFs due to low tumor content or subclonal heterogeneity, or harbor non-V600 mutations. Here, we show that Sanger sequencing using the NuProbe VarTrace BRAF assay, based on the Blocker Displacement Amplification (BDA) technology, is capable of detecting BRAF V600 mutations down to 0.20% VAF from FFPE lymph node tissue samples. Comparison experiments on adjacent tissue sections using BDA Sanger, immunohistochemistry (IHC), digital droplet PCR (ddPCR), and NGS showed 100% concordance among all 4 methods for samples with BRAF mutations at ≥ 1% VAF, though ddPCR did not distinguish the V600K mutation from the V600E mutation. BDA Sanger, ddPCR, and NGS (with orthogonal confirmation) were also pairwise concordant for lower VAF mutations down to 0.26% VAF, but IHC produced a false negative. Thus, we have shown that Sanger sequencing can be effective for rapid detection and quantitation of multiple low VAF BRAF mutations from FFPE samples. BDA Sanger method also enabled detection and quantitation of less frequent, potentially actionable non-V600 mutations as demonstrated by synthetic samples.

scholarly journals Targeted Next Generation Sequencing Identifies Novel Genetic Mutations in Patients with Waldenstrom's Macroglobulinemia/Lymphoplasmacytic Lymphoma or IgM-Monoclonal Gammopathies of Undetermined Significance

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2928-2928 ◽  
Author(s):  
Marzia Varettoni ◽  
Silvia Zibellini ◽  
Ettore Rizzo ◽  
Luca Malcovati ◽  
Virginia Valeria Ferretti ◽  
...  
Keyword(s):  
Sanger Sequencing ◽  
Somatic Mutations ◽  
Genetic Mutations ◽  
Wild Type ◽  
Targeted Next Generation Sequencing ◽  
Coefficient Of Concordance ◽  
Generation Sequencing ◽  
Myd88 L265p ◽  
Undetermined Significance

Abstract Background. TheMYD88 (L265P) somatic mutation is present in more than 90% of patients (pts) with Waldenström's Macroglobulinemia (WM)/lymphoplasmacitic lymphoma (LPL). The second most common mutations are nonsense (NS) or frameshift (FS) mutations in the CXCR4 gene, detectable in approximately 25-30% of WM pts by Sanger sequencing. Limited data are available about other genetic mutations in WM/LPL and its precursor condition IgM-monoclonal gammopathy of undetermined significance (IgM-MGUS). Pts and methods. Using targeted next generation sequencing (NGS), we evaluated the prevalence of somatic mutations of 11 genes selected on the basis of evidences available from the literature (MYD88, CXCR4, ARID1A, KMT2D, TP53, NOTCH2, PRDM1, CD79b, TRAF3,TNFAIP3, MYDBBP1A) in 119 pts, classified as WM/LPL (n=63) or IgM-MGUS (n=56) according to International Consensus Criteria. Median age of pts (67 males, 52 females) was 65 years (range: 38-82). Samples were collected at diagnosis (n=101), after diagnosis but before any treatment (n=9) or at progression after therapy (n=9). Paired tumor and germline DNA extracted respectively from CD19-selected and CD19-depleted bone marrow (BM) mononuclear cells was available in all pts. Mean resequencing depth across gene panel was 1009x. Only mutations tagged as oncogenic or possibly oncogenic based on information derived from the literature and on in silico prediction effect were considered in the analysis. For MYD88 (L265P) and CXCR4 mutations, results obtained with NGS were compared with those obtained respectively with allele-specific PCR (AS-PCR) and Sanger sequencing. Results. Overall, we found 151 mutations in 88 pts (74%). The median number of mutations was significantly higher in WM/LPL as compared with IgM-MGUS and in pts previously treated as compared with untreated ones (median 2 versus 1, P < 0.001 for both comparisons). MYD88 mutations were found in 80/119 pts (67%), with a median allele burden of 34.2% (range: 2.5-93.3%). The prevalence of MYD88 mutations was significantly higher in WM/LPL as compared with IgM-MGUS (86% versus 46%, P <0.001). MYD88 mutations other than classical L265P (n=76) were found in 4 pts and were represented by V217F (n=2), S219C (n=1), M232T (n=1). Fifteen pts who were MYD88 (L265P) wild-type by NGS were found to be mutated by AS-PCR (K coefficient of concordance between NGS and AS-PCR: 70%, P < 0.001). CXCR4 mutations were found in 19/119 pts (16%), with a median allele burden of 34% (range: 4.2-84%). The prevalence of CXCR4 mutations was significantly higher in WM/LPL as compared with IgM-MGUS (24% versus 7%, P < 0.02). The K coefficient of concordance between NGS and Sanger was 83% (P < 0.001), with 2 pts mutated only by NGS and 2 pts mutated only by Sanger. Somatic mutations were also found in KMT2D (formerly known as MLL2) (16% of pts), TP53 (8%), NOTCH2 (7%), PRDM1 (4%), ARID1A (3%), CD79b (2%), and TRAF3 (1%). No mutations were found in MYBBP1A and TNFAI3. Overall, the prevalence of these mutations was significantly lower in pts wild-type either for MYD88 or CXCR4 as compared with those with MYD88 and/or CXCR4 mutations (15% versus 41%, P = 0.04). The prevalence of KMT2D mutations was significantly higher in WM/LPL as compared with MGUS (25% versus 5%), while for the other genes the distribution was not statistically different according to diagnosis. With a median follow-up of 20 months (range: 0-264), we did not find a statistically significant correlation between genetic mutations and pts' outcome in terms of overall survival or time to first treatment. Conclusions. In this cohort of pts with WM/LPL and IgM-MGUS studied with NGS we could demonstrate that: i) NGS identifies MYD88 mutations other than L265P in a small proportion of pts; ii) the prevalence of CXCR4 mutations by Sanger is confirmed by NGS, despite the higher sensitivity of the latter method; iii) the subgroup of pts wild type either for MYD88 or CXCR4 shows a low incidence of other genetic mutations; iv) 25% of pts with WM/LPL were found to carry KMT2D mutations, a prevalence similar to that reported in marginal zone lymphoma; v) genetic mutations are more common in WM/LPL than in IgM-MGUS in agreement with the hypothesis that multiple genetic hits are required for progression from a pre-benign condition to a neoplastic disease; vi) due to the indolent nature of these disorders, longer follow-up is probably needed to see the prognostic impact of these mutations, if any. Disclosures No relevant conflicts of interest to declare.

scholarly journals Fluorescence Polarization Immunoassay for Determination of Enrofloxacin in Pork Liver and Chicken

Molecules ◽  
2019 ◽  
Vol 24 (24) ◽  
pp. 4462
Author(s):  
Xing Shen ◽  
Jiahong Chen ◽  
Shuwei Lv ◽  
Xiulan Sun ◽  
Boris B. Dzantiev ◽  
...  
Keyword(s):  
Fluorescence Polarization ◽  
Limit Of Detection ◽  
Polyclonal Antibodies ◽  
Screening Method ◽  
Sample Pretreatment ◽  
High Sensitivity ◽  
Cross Reactivity ◽  
Fluorescence Polarization Immunoassay ◽  
Pork Liver ◽  
Sensitivity Specificity

Enrofloxacin (ENR) is a widely used fluoroquinolone (FQ) antibiotic for antibacterial treatment of edible animal. In this study, a rapid and highly specific fluorescence polarization immunoassay (FPIA) was developed for monitoring ENR residues in animal foods. First, ENR was covalently coupled to bovine serum albumin (BSA) to produce specific polyclonal antibodies (pAbs). Three fluorescein-labeled ENR tracers (A, B, and C) with different spacers were synthesized and compared to obtain higher sensitivity. Tracer C with the longest arm showed the best sensitivity among the three tracers. The developed FPIA method showed an IC50 (50% inhibitory concentration) of 21.49 ng·mL−1 with a dynamic working range (IC20–IC80) of 4.30–107.46 ng·mL−1 and a limit of detection (LOD, IC10) of 1.68 ng·mL−1. The cross-reactivity (CR) of several structurally related compounds was less than 2%. The recoveries of spiked pork liver and chicken samples varied from 91.3% to 112.9%, and the average coefficients of variation were less than 3.83% and 5.13%, respectively. The immunoassay took only 8 min excluding sample pretreatment. This indicated that the established method had high sensitivity, specificity, and the advantages of simplicity. Therefore, the proposed FPIA provided a useful screening method for the rapid detection of ENR residues in pork liver and chicken.

Development of an Ultra-Deep Sequencing Method on Ion Torrent PGM to Detect Mutations in a Panel of Genes Relevant to Disease Progression and Drug Resistance in Chronic Lymphocytic Leukaemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5634-5634
Author(s):  
Sozan Karim
Keyword(s):  
Drug Resistance ◽  
Next Generation Sequencing ◽  
Disease Progression ◽  
Sanger Sequencing ◽  
Lymphocytic Leukaemia ◽  
Ion Torrent ◽  
Coverage Depth ◽  
Ion Torrent Pgm ◽  
Sequencing Method ◽  
Generation Sequencing

Abstract Chronic lymphocytic leukaemia (CLL) is the most common adult leukaemia in the Western world. The disease is characterised by a great variability in clinical course and response to treatment. Acquired somatic mutations in genes related to multiple signal pathways have recently emerged as important factors contributing to this heterogeneity. These mutations accumulate with disease progression or development of therapy resistance. However, with conventional methods they are very difficult to be detected in a single test, particularly when the size of the affected cell population is small. Therefore, there is a clinical need for a rapid, reliable, affordable and highly sensitive sequencing method to monitor these mutations from an early stage of this disease. Herein we developed an ultra-deep next generation sequencing approach based on Ion Torrent PGM to sequence a total of 246 exons of 15 genes including TP53, ATM, BIRC3, NOTCH1, SF3B1, MYD88, LRP1B, SAMHD1, FBXW7, POT1, HISTIH1E, XPO1, CHD2, PCLO and ZFPM2. These genes were selected because mutations in them have been reported to involve at least 5% of CLL patients and associate with poor outcome of CLL. Serial CLL samples were collected at multiple time points from diagnosis to disease progression/drug resistance from a cohort of 33 patients. Initially, target DNA in samples taken at advanced disease stages, without or with mutations in TP53 as detected and quantified by FASAY assay and Sanger sequencing, was enriched and amplified using Agilent HaloPlex probes, with 4604 amplicons for a sequenceable size of 135.91 Kbp of each sample. On each Ion 318 Chip (1 GB output), barcoded DNA enrichment preparations from 4 patients were sequenced by the PGM. For confirmation, the same patient samples were tested repeatedly in an independent experiment starting from DNA preparation. The sequence data were aligned to a human reference genome (Hg19); variants were called by the Torrent Variant Caller (v4.0-6) and visualised with the IGV. Results showed that the target coverage was 99.999% and an average coverage depth 3941x. Importantly, all of the known TP53 mutations were repeatedly detected at expected frequencies. In addition, extra mutations at lower levels (5% - 20% alleles) in multiple genes, including TP53 and SF3B1, were detected. In a sensitivity test of this method, DNA samples from 5 cases, each with a known % of mutation in TP53measured by FASAY assay and Sanger sequencing, were pooled together and then mixed with a wild-type DNA sample to serially dilute these mutations to 20% - 0.2% alleles before target enrichment. With an average coverage depth of 1843x (range: 1610 - 2187) for these 5 target locations, each mutation was readily detected at 20%, 5% and 1%, with the average quality scores being 1497.3, 61.2 and 29.4, respectively. However, only 3 out the 5 mutations could be detected at the level of 0.2%. Taken together, we have successfully developed a sensitive next generation sequencing method for detecting mutations in a CLL gene panel. This allows us to monitor multiple mutations affecting as low as 1% of alleles in CLL samples throughout the disease course and possibly discover clinically useful biomarker(s) for CLL progression and resistance to therapy. Disclosures No relevant conflicts of interest to declare.

Analysis of correlation between oncogene mutation and response to chemotherapy in all RAS wild type metastatic colorectal cancer, using next-generation sequencing technology.

2015 ◽  
Vol 33 (3_suppl) ◽  
pp. 553-553
Author(s):  
Akio Higuchi ◽  
Rika Kasajima ◽  
Manabu Shiozawa ◽  
Masahiro Asari ◽  
Masaaki Murakawa ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Next Generation Sequencing ◽  
Driver Mutations ◽  
Wild Type ◽  
Sequencing Technology ◽  
Mutation Status ◽  
Next Generation Sequencing Technology ◽  
Response Group ◽  
Generation Sequencing ◽  
Kras Mutation Status

553 Background: Targeted therapies of monoclonal antibodies have changed the treatment of metastatic colorectal cancer (mCRC). A target therapy with chemotherapy regimen for mCRC was decided by KRAS mutation status (KRAS exon2 [codon12, codon13]). Currently, there are many reports suggesting that in addition to analysis of KRAS mutation status, the evaluation of EGFR gene copy number, levels of EGFR ligands, BRAF, NRAS, PIK3CA mutations could be helpful to have a more accurate selection of patients who may have a benefit from anti-EGFR targeted drugs. Methods: Mutation status of 50 oncogenes were analysed in 35 mCRC patients with all RAS wild type, using next-generation sequencing technology. The response for chemotherapy was classified response group (R group) and non-response group (N group) by RECIST. The relation between mutation status of 50 oncogenes and the response for chemotherapy was assessed. Results: There were 25 oncogene mutations in the 50 genes. Driver mutation associated with oncogenic mutation deeply were 5 oncogenes, which were PIK3CA, AKT1, BRAF, PDGFRA and TP53. Only BRAF mutation was significantly associated with poor chemo response in the 5 oncogenes. A case which had two driver mutations was only in the N group. One of the two driver mutations was tumor suppressor gene, TP53. Conclusions: BRAF mutation and the number of driver mutations are key predictors of chemosensitivity in the mCRC cases with all RAS wild type.

scholarly journals High Sensitivity Sanger Sequencing Detection of BRAF Mutations in Metastatic Melanoma FFPE Tissue Specimens

2020 ◽  
Author(s):  
Lauren Cheng ◽  
Lauren Haydu ◽  
Ping Song ◽  
Jianyi Nie ◽  
Michael Tetzlaff ◽  
...  
Keyword(s):  
Sanger Sequencing ◽  
Limit Of Detection ◽  
False Negative ◽  
High Sensitivity ◽  
Ffpe Tissue ◽  
Braf Mutations ◽  
Tissue Samples ◽  
Ffpe Samples ◽  
Droplet Pcr ◽  
Tissue Specimens

Abstract Mutations in the BRAF gene at or near the p. V600 locus are informative for therapy selection, but current methods for analyzing FFPE tissue DNA generally have a limit of detection of 5% variant allele frequency (VAF), or are limited to the single variant (V600E). These can result in false negatives for samples with low VAFs due to low tumor content or subclonal heterogeneity, or harbor non-V600 mutations. Here, we show that Sanger sequencing using the NuProbe VarTraceTM BRAF assay, based on the Blocker Displacement Amplification (BDA) technology, is capable of detecting BRAF V600 mutations down to 0.26% VAF from FFPE lymph node tissue samples. Comparison experiments on adjacent tissue sections using BDA Sanger, immunohistochemistry (IHC), digital droplet PCR (ddPCR), and NGS showed 100% concordance among all 4 methods for samples with BRAF mutations at ≥1% VAF, though ddPCR did not distinguish the V600K mutation from the V600E mutation. BDA Sanger, ddPCR, and NGS (with orthogonal confirmation) were also pairwise concordant for lower VAF mutations down to 0.26% VAF, but IHC produced a false negative. Thus, we have shown that Sanger sequencing can be effective for rapid detection and quantitation of multiple low VAF BRAF mutations from FFPE samples. BDA Sanger method also enabled detection and quantitation of less frequent, potentially actionable non-V600 mutations as demonstrated by synthetic samples.

Rapid detection of non-deletional mutations causing α-thalassemia by multicolor melting curve analysis

2016 ◽  
Vol 54 (3) ◽  
Author(s):  
Qiuying Huang ◽  
Xudong Wang ◽  
Ning Tang ◽  
Chunjiang Zhu ◽  
Tizhen Yan ◽  
...  
Keyword(s):  
Rapid Detection ◽  
Sanger Sequencing ◽  
Melting Curve ◽  
High Sensitivity ◽  
Cost Effective ◽  
Curve Analysis ◽  
Melting Curve Analysis ◽  
Sensitivity Specificity

AbstractThe assay consisted of one pair of primers specific for theThe assay had a reproducibility of 100%, could detect gDNA of different genotype as low as 1 ng per reaction, and had an overall accuracy of 100% when compared with RDB analysis and Sanger sequencing.The developed assay is rapid, robust, and cost-effective while maintaining high sensitivity, specificity, and throughput.

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