Sequencing-based variant detection in the polyploid crop oilseed rape

A single acquired mutation in the JAK2 gene has recently been described in human myeloproliferative disorders, including most patients with polycythemia vera and about half of those with essential thrombocythemia and idiopathic myelofibrosis. Reliable and easily implemented methods for detection of this V617F mutation promise to revolutionize the way these disorders are diagnosed and classified, and may in the future have implications for targeted therapeutics. Two polymerase chain reaction-based methods for detection of the mutation are described here. One method is based on allele-specific amplification of the mutant band, and the other on elimination of a restriction enzyme recognition sequence by the mutation. Both methods are significantly more sensitive than conventional sequencing techniques, and could be readily implemented in a molecular diagnostic laboratory.

Robust and Sensitive Detection of Insertions, Deletions and Point Mutations In CEBPA, a GC-Rich Content Gene, Using 454 Next-Generation Deep-Sequencing (NGS).

The diagnostic hallmark of the dystrophic forms of epidermolysis bullosa (DEB), a group of heritable blistering skin diseases, is abnormalities in the anchoring fibrils at the dermal-epidermal basement membrane zone. Since type VII collagen is the major, if not the exclusive, component of the anchoring fibrils, the corresponding gene (COL7A1) is the candidate gene in DEB. Recent cloning of the type VII collagen cDNA and elucidation of the exon-intron organization of the gene have provided the basis for us to develop a novel strategy for identification of sequence variants in COL7A1. Optimization of 72 balanced primer pairs corresponding to flanking intronic sequences allowed PCR amplification of all 118 exons directly from genomic DNA. The PCR products were examined by heteroduplex analysis followed by comparative nucleotide sequencing. More than 100 sequence variants have been identified thus far in COL7A1 using this method, some of which are single base pair polymorphisms and many of which are pathogenetic mutations contributing to the blistering phenotype in DEB. The comprehensive method described is useful for rapid, reliable, and sensitive detection of sequence variants in COL7A1. We demonstrate the utility of this novel strategy in mutation detection and prenatal exclusion of RDEB in a consanguineous family at risk for recurrence. Hum Mutat 10:408–414, 1997. © 1997 Wiley-Liss, Inc.

Sensitive and quantitative detection of mutations associated with clinical resistance to STI-571

Detection of the Single Hotspot Mutation in the JH2 Pseudokinase Domain of Janus Kinase 2 in Bone Marrow Trephine Biopsies Derived from Chronic Myeloproliferative Disorders

A major question for the Alberta oilsands extraction industry is how to return tailings ponds to their original ecosystem functions at the end of their lifespan. This question is one of the most pressing in the oil industry, and while many potential solutions are being trialed, none are without issue. One solution is the creation of end pit lakes (EPLs), formed by filling a mine pit or tailings pond with fluid tailings and covering this with a freshwater cap. Over time, tailings settle to the bottom of the lake and become sequestered. As of 2018, there were 23 plans to pursue EPLs in Albertan oilsands mining operations (COSIA 2021), so research in oilsands reclamation via EPLs is essential. Alberta has only one full-scale EPL formed using tailings, Base Mine Lake (BML). It has been under reclamation since 2011. Although BML water quality is gradually improving over time, one persistent problem is the production of methane bubbles from the entrenched hydrocarbons. Ebullition of these bubbles carries contaminants into the water column. The potential to limit methane production biologically by stimulating methanotroph metabolism, is therefore of interest. Methanotrophs are microbes that consume methane. These methanotrophs can be aerobic, in the water column, or anaerobic below the sediment interface, where oxygen is depleted. Anaerobic oxidizers of methane, or AOM, are of particular interest due to methane only being produced in anaerobic conditions. However, AOM are not well-studied and there are many gaps in our knowledge about them. Base Mine Lake presents an important opportunity to document the presence of AOM in oilsands ecosystems, and identify if AOM can remove methane before it enters the water column. Methane is probably not the limiting factor for AOM in BML- instead, the electron acceptors used in the absence of oxygen are scarce. If microbiological methane removal were to increase after supplementing electron acceptors such as nitrate, sulfate, or iron, then this could be applied to the reclamation of artificial end pit lake systems. Our research aims to illustrate if AOM are present in BML, and if we can stimulate their metabolism via electron acceptor amendment. We performed amplicon and metagenomic sequencing across 5 sediment cores from BML. These cores penetrate over two meters below the water-sediment interface, up to 14 meters below the water’s surface. We designed anaerobic, 13C-isotope-spiked incubations for this sediment to track if any CH4 to CO2 conversion was underway. By tagging methane molecules with 13C, we are able to follow the biogeochemical transformations and demonstrate if any 13CH4 is converted to 13CO2, which would indicate methanotrophic activity. Additionally, microbes who take up 13C produce heavier DNA, possibly allowing us to identify the DNA of the players at work. Additionally, Illumina sequencing was done on all incubations to compare how electron acceptor addition prompted community shifts. In the incubation experiments, 13CO2 is being produced in several core samples incubated without O2. This finding indicates that methanotrophs are, in some capacity, active in anoxic conditions. When comparing different amendments of electron acceptors, there did appear to be a significant amount (p = 0.027) of 13CO2 produced in the sulfate-added 13CH4 treatment (Fig. 1). Other amendments had encouraging results, but were not statistically significant. Ongoing trials will provide more illuminating results via larger sample sizes, which will be completed by the end of the summer. Next we will characterize communities in the post-incubation sediment, using both amplicon sequencing and DNA stable isotope probing. This will suggest not only the identity of the organisms at play, but will also illuminate which electron acceptors were most beneficial to which groups. This analysis is complex and underway. Overall, the study of methanotrophy in artificial ecosystems with high emissions is an area of great interest, due to the high potential for amendment and improvement of the ecosystems using methanotrophs, as well as the ongoing discovery on AOM in artificial ecosystems. Oilsands end pit lakes are of interest, as an issue of cultural and political emphasis.

Digital PCR detection of EGFR somatic mutations in non-small-cell lung cancer formalin fixed paraffin embedded samples

Nucleophosmin (NPM1) mutation is one of the most common recurring genetic abnormalities seen in acute myeloid leukemia (AML). Immunohistochemistry serves as a cost effective and simple surrogate testing method for detection of NPM1 mutation. This study was conducted to evaluate the frequency of aberrant cytoplasmic nucleophosmin 1 expression in leukemic blast cells on formalin fixed bone marrow trephine biopsy (BMB) sections and also to correlate this data with the reference molecular method (reverse transcriptase-polymerase chain reaction; RT-PCR and gene sequencing), where available. Immunostains were performed using mouse anti-NPM1 monoclonal antibody on 71 paraffin embedded bone marrow biopsies (BMB) of patients with AML of any French-American-British (FAB) subtype. Results of immunohistochemistry (IHC) were then compared with the reference molecular method. The proportion of NPM1 expression by immunostaining in AML cases was found to be 17%. Twelve of the total 71 cases demonstrated cytoplasmic nucleophosmin (NPMc+) on immunostaining. Eleven of the positive cases that were correlated with the molecular standard demonstrated mutation in exon 12 of NPM1 gene. Cytoplasmic nucleophosmin expression by immunostaining was found to be in complete agreement with the standard molecular method. In a resource restricted setup, the information from this study might help in providing an inexpensive and accurate detection method to facilitate introduction of this marker in diagnostic and prognostic workup of AML especially in patients showing normal karyotype and no common recurrent translocations.

Among patients with colorectal cancer (CRC), KRAS mutations were reported to occur in 30-51 % of all cases. CRC patients with KRAS mutations were reported to be non-responsive to anti-epidermal growth factor receptor (EGFR) monoclonal antibody (MoAb) treatment in many clinical trials. Hence, accurate detection of KRAS mutations would be critical in guiding the use of anti-EGFR MoAb therapies in CRC. In this study, we carried out a detailed investigation of the efficacy of a wild-type (WT) blocking real-time polymerase chain reaction (PCR), employing WT KRAS locked nucleic acid blockers, and Sanger sequencing, for KRAS mutation detection in rare cells. Analyses were first conducted on cell lines to optimize the assay protocol which was subsequently applied to peripheral blood and tissue samples from patients with CRC. The optimized assay provided a superior sensitivity enabling detection of as little as two cells with mutated KRAS in the background of 10(4) WT cells (0.02 %). The feasibility of this assay was further investigated to assess the KRAS status of 45 colorectal tissue samples, which had been tested previously, using a conventional PCR sequencing approach. The analysis showed a mutational discordance between these two methods in 4 of 18 WT cases. Our results present a simple, effective, and robust method for KRAS mutation detection in both paraffin embedded tissues and circulating tumour cells, at single-cell level. The method greatly enhances the detection sensitivity and alleviates the need of exhaustively removing co-enriched contaminating lymphocytes.

The new massively parallel sequencing methods are so astonishing one wonders whether space aliens are secretly behind them. One technician, running a single instrument, can obtain up to ~1 billion bases of DNA sequence in a few days. Here we describe the new sequencing methods, briefly present a few applications in HIV research, and then speculate on future directions.

Elevator button (EB) detection is a very important research issue in home service robotics area, this paper describes an effective method for EB detection with mirroring effects in completely reflective surfaces. Line features and other local features (such as corner, SIFT and color, etc) are extracted from stereo images to represent the EBs, since the EBs are almost in the complete reflective surface with mirror effect, so there are many ¿virtual¿ features, which are not in the plane of EB panel, in order to get only ¿real¿ features from the EB panel, homography matrix between two stereo images is applied to eliminate the ¿virtual¿ features effectively. After feature matching with the EB model, multiple interpretations with corresponding weights and uncertainties are generated as hypothesis of EBs position. Finally, the proposed particle filtering based interpretation fusion algorithm is applied to estimate the multiple EBs position robustly and sequentially. Experimental results in the real environment demonstrate that robust detection and convergence performance are achieved when confronted by serious mirror effects.

Background The deregulation of the PI3K/AKT signaling pathway is essential to malignant cellular processes of breast cancer, including proliferation, apoptosis, and drug response. Oncogenic activating somatic mutations in the PI3K/AKT pathway are pervasive. However, it remains difficult to discriminate between driver and passenger mutations. This study was conducted to identify the landscape of genetic mutations in the PI3K/AKT pathway using Amplicon Sequencing in a Chinese population. Notably, we developed a Gateway-based mutation barcoding (GaMB) library which enables a high-throughput mutation-phenotype screen for specific vulnerable mutations that contribute to the cancer development and drug resistance. Method We collected 149 breast cancer specimens in a Chinese population and performed Ion Torrent Amplicon Sequencing for the key genes in PI3K/AKT pathway: PIK3CA, PIK3R1, AKT1, AKT2, AKT3, PTEN, PDK1, and the canonical tumor suppressor gene TP53, at 1000× coverage. Next, we established a high-throughput GaMB library that contained all of the PIK3CA mutations, either newly identified in Chinese population or reported in TCGA and COSMIC database, and tagged each mutations with a specific barcode. We then applied this library to functional screening processes using proliferation and drug response selection (doxorubicin or BKM-120) assays through which we screened the functional mutations with specific characteristics. The genomic DNA of the pooled surviving cells from the library, as well as the original cells before the screenings, was extracted and used for PCR amplification of the barcode regions, and then detected using Illumina Miseq sequencing to analyze the functional mutations. We then validated the cellular 2D- & 3D- proliferation abilities and the status of PI3K/AKT pathway activation in presence of identified mutations, respectively. Result Mutations in the PIK3CA (44%), PIK3R1 (37%), AKT3 (15%) and PTEN (12%) genes were the most prevalent. Mutations in PIK3CA were present in 65 samples (43.6%) which is similar to that reported in TCGA database. PIK3R1 (37%) was found significantly mutated, with a novel recurrent mutation, N595S, being identified in 24 patients. Similarly, AKT1 (10.1%), AKT2 (10.1%), and AKT3 (14.8%) mutations were present at a higher frequency in our population than has been reported in the TCGA and COSMIC database. In the PIK3CA-GaMB library, our highest-ranking mutations included the previously validated deleterious mutations H1047R and E545K and several mutations of uncertain significance, including E39K, G1049R, N345I, N345K, M1043V, and H1047T. In the validation assays, we found a high phenotype-consistency of these identified mutations using these functional validation techniques. The breast cancer cells harboring identified mutations exhibit a relatively higher proliferation ability and tolerance to chemotherapy and pathway inhibitors. Conclusion This study identified the landscape of genetic mutations in the PI3K/AKT pathway using Amplicon Sequencing in a Chinese population. A novel developed GaMB screening platform may allow the rapid identification of significant mutations that dominate breast cancer development and drug responses during treatment. Citation Format: Chen L, Yang L, Yao L, Hu X, Shao Z. The mutation detection and a high throughput screening of driver mutations in PI3K/AKT pathway based on next generation sequencing [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-07-13.

Improved Methodology for Detection of Mutations in the BCR-ABL Fusion Gene That Cause Resistance to the Tyrosine Kinase Inhibitor Imatinib.

Digital PCR (dPCR) is being increasingly used for the quantification of sequence variations, including single nucleotide polymorphisms (SNPs), due to its high accuracy and precision in comparison with techniques such as quantitative PCR (qPCR) and melt curve analysis. To develop and evaluate dPCR for SNP detection using DNA, RNA, and clinical samples, an influenza virus model of resistance to oseltamivir (Tamiflu) was used. First, this study was able to recognize and reduce off-target amplification in dPCR quantification, thereby enabling technical sensitivities down to 0.1% SNP abundance at a range of template concentrations, a 50-fold improvement on the qPCR assay used routinely in the clinic. Second, a method was developed for determining the false-positive rate (background) signal. Finally, comparison of dPCR with qPCR results on clinical samples demonstrated the potential impact dPCR could have on clinical research and patient management by earlier (trace) detection of rare drug-resistant sequence variants. Ultimately this could reduce the quantity of ineffective drugs taken and facilitate early switching to alternative medication when available. In the short term such methods could advance our understanding of microbial dynamics and therapeutic responses in a range of infectious diseases such as HIV, viral hepatitis, and tuberculosis. Furthermore, the findings presented here are directly relevant to other diagnostic areas, such as the detection of rare SNPs in malignancy, monitoring of graft rejection, and fetal screening.

BackgroundWhile massively parallel DNA sequencing methods continue to evolve rapidly, the benchmark technique for detection and verification of rare (particularly disease-causing) sequence variants remains four-colour dye-terminator sequencing by capillary electrophoresis....