Ion Semiconductor Research Articles

Next generation sequencing as a suitable analysis for authentication of degraded and ancient DNA

Ancient DNA studies require the authentication of the results since it is essential to verify the absence of contaminating sequences. Cloning the amplified products obtained from these samples has been one of the principal methods traditionally used to assure the absence of contamination. However, this is an arduous analysis, not routinely applied in forensic laboratories. Recently, the development of next generation sequencing (NGS) technologies opens new ways to authenticate ancient DNA results. NGS data allow the discrimination between endogenous and contaminating sequences. Here we present the results obtained by both traditional cloning and next generation sequencing based on ion semiconductor sequencing on Ion Torrent™ PGM®. The comparison of both methods shows that ion semiconductor sequencing provides a better estimation of the proportion of contaminating sequences present in the sample and more information about the preservation state of the analyzed DNA than traditional cloning. Thus, NGS technology, and in particular, ion semiconductor sequencing, is revealed as a suitable method to authenticate the results obtained from degraded and ancient DNA samples since it provides more information, it is less time and money consuming and it would be unaffordable to obtain the same quantity of data by traditional cloning.

Validation of the Ion Torrent PGM sequencing for the prospective routine molecular diagnostic of colorectal cancer

ObjectivesTreatment individualization based on specific molecular biomarkers is becoming increasingly important in oncology. In colorectal cancer (CRC), the molecular characterization of RAS and BRAF mutation status for prognostic and predictive purposes is commonly performed by different validated methods. However, as the number of clinically relevant mutations to be analyzed increases, the definition of new approaches for more sensitive, rapid and economic patient selection urges. To this aim, we evaluated the Ion Semiconductor sequencing using the Ion Torrent Personal Genome Machine (IT-PGM) in our routine molecular diagnostics for CRC in comparison with the gold standard direct Sanger sequencing. Design and methodsFormalin-fixed and paraffin-embedded tumor tissues obtained by surgery or biopsy of 66 CRCs were collected. DNA was extracted and sequenced by IT-PGM and Sanger method. ResultsThe proposed IT-PGM sequencing strategy exceeded the 500 reads of coverage for all clinically relevant RAS/BRAF amplicons in most samples and thus guaranteed optimal determination. Indeed, the frequencies and the mutational spectrum of RAS and BRAF mutations were in agreement with literature data and revealed 100% concordance between the IT-PGM and routine Sanger sequencing approaches. Turnaround time and cost evaluation indicate that the IT-PGM sequencing permits the characterization of the clinically relevant mutational spots at lower cost and turnaround time compared to Sanger sequencing and allows inclusion of additional amplicons whose characterization may acquire significance in the very next future. ConclusionThe IT-PGM is a valid, flexible, sensitive and economical method alternative to the Sanger sequencing in routine diagnostics to select patients for anti-epidermal growth factor receptor therapy for metastatic CRC.

Circulating tumor cells in hepatocellular carcinoma: a pilot study of detection, enumeration, and next-generation sequencing in cases and controls

BackgroundCirculating biomarkers are urgently needed in hepatocellular carcinoma (HCC). The aims of this study were to determine the feasibility of detecting and isolating circulating tumor cells (CTCs) in HCC patients using enrichment for epithelial cell adhesion molecule (EpCAM) expression, to examine their prognostic value, and to explore CTC-based DNA sequencing in metastatic HCC patients compared to a control cohort with non-malignant liver diseases (NMLD).MethodsWhole blood was obtained from patients with metastatic HCC or NMLD. CTCs were enumerated by CellSearch then purified by immunomagnetic EpCAM enrichment and fluorescence-activated cell sorting. Targeted ion semiconductor sequencing was performed on whole genome-amplified DNA from CTCs, tumor specimens, and peripheral blood mononuclear cells (PBMC) when available.ResultsTwenty HCC and 10 NMLD patients enrolled. CTCs ≥ 2/7.5 mL were detected in 7/20 (35%, 95% confidence interval: 12%, 60%) HCC and 0/9 eligible NMLD (p = 0.04). CTCs ≥ 1/7.5 mL was associated with alpha-fetoprotein ≥ 400 ng/mL (p = 0.008) and vascular invasion (p = 0.009). Sequencing of CTC DNA identified characteristic HCC mutations. The proportion with ≥ 100x coverage depth was lower in CTCs (43%) than tumor or PBMC (87%) (p < 0.025). Low frequency variants were higher in CTCs (p < 0.001).ConclusionsCTCs are detectable by EpCAM enrichment in metastatic HCC, without confounding false positive background from NMLD. CTC detection was associated with poor prognostic factors. Sequencing of CTC DNA identified known HCC mutations but more low-frequency variants and lower coverage depth than FFPE or PBMC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1195-z) contains supplementary material, which is available to authorized users.

Translating sanger-based routine DNA diagnostics into generic massive parallel ion semiconductor sequencing.

Dideoxy-based chain termination sequencing developed by Sanger is the gold standard sequencing approach and allows clinical diagnostics of disorders with relatively low genetic heterogeneity. Recently, new next generation sequencing (NGS) technologies have found their way into diagnostic laboratories, enabling the sequencing of large targeted gene panels or exomes. The development of benchtop NGS instruments now allows the analysis of single genes or small gene panels, making these platforms increasingly competitive with Sanger sequencing. We developed a generic automated ion semiconductor sequencing work flow that can be used in a clinical setting and can serve as a substitute for Sanger sequencing. Standard amplicon-based enrichment remained identical to PCR for Sanger sequencing. A novel postenrichment pooling strategy was developed, limiting the number of library preparations and reducing sequencing costs up to 70% compared to Sanger sequencing. A total of 1224 known pathogenic variants were analyzed, yielding an analytical sensitivity of 99.92% and specificity of 99.99%. In a second experiment, a total of 100 patient-derived DNA samples were analyzed using a blind analysis. The results showed an analytical sensitivity of 99.60% and specificity of 99.98%, comparable to Sanger sequencing. Ion semiconductor sequencing can be a first choice mutation scanning technique, independent of the genes analyzed.

A Novel Method for the Detection of Minimal Residual Disease in B-Cell Malignancies: Ion Semiconductor Sequencing for the Evaluation of Igh Gene Rearrangements

Background:Minimal residual disease (MRD) detection is of high clinical relevance in patients with B-cell malignancies and is generally a surrogate parameter to evaluate treatment response and long-term prognosis. IgH gene rearrangements can be used as molecular marker in approximately 80% of lymphoma and myeloma patients since they represent lineage-specific markers and the complementarity determining region 3 (CDR3) is unique to each clone. To date, allele specific oligonucleotide polymerase chain reaction (ASO-PCR) and real-time quantitative polymerase chain reaction (RQ-PCR) are considered the most sensitive and widely applicable methods for MRD detection. A major disadvantage of ASO-PCR and RQ-PCR assays, is the use of specific primers and probes for every individual patient. Clone-specific primers and probes are not only expensive but also time-consuming to design and to test, which limits their wide applicability in the clinical setting. The recent major improvements in next generation sequencing (NGS) technologies, provide the opportunity to identify and quantify clonotypes with consensus primers combining the benefits of high sensitivity and universal applicability. The present work was designed to overcome ASO-PCR and RQ-PCR limitations by developing a feasible method for rearranged IgH genes amplification, NGS and analysis using Ion Torrent Personal Genome Machine (IT-PGM).Methods:To define a multiplex PCR protocol, DNA from 7 CLL patients, previously shown to bare a family specific clonal VDJ rearrangement, was amplified with a pool of the seven different family-specific IgH-V primers, and a consensus JH primer (Voena et al., Leukemia 1997). After Sanger sequencing, results were compared to the ones obtained with singleplex PCR protocol. Once validated, the multiplex PCR protocol was used to amplify DNA from patients and serially diluted (up to 10-8 ) DNA from Namalwa cell line (bearing a known IgH rearrangement) and subsequently sequenced on the IT-PGM using the 316 Ion-chip. NGS data were analyzed by using the IMGT-High V-Quest web server tool and the statistical software R. RQ-PCR was used to quantify the specific VDJ rearrangement in the serially diluted Namalwa DNA solutions and in DNA from patients as previously described (Farina et al, Haematologica 2009). RQ-PCR data were analyzed through a relative quantification procedure.Results:The multiplex PCR reactions we have tested, demonstrated the same specificity as the standard singleplex PCR protocol and therefore was used to construct the DNA library required for IT-PGM-based sequencing. The IT-PGM sequencing output is represented by at least 400000 reads per sample with a minimum average coverage of the VDJ repertoire of 500x. The IMGT-High V-quest tool allows a user-friendly web based analysis and a deep molecular characterization of the IgH recombinatorial repertoire. Namalwa clonal CRD3 sequences were detected up to a dilution of 10-5 without the need for specific CDR3 primers. Comparability of NGS and ASO RQ-PCR results was assessed. The use of CDR3 specific primers, along with the specific IgH-V family fluorescent probe, enabled the identification of clonal VDJ rearrangements with a sensitivity up to 10-5 (2/3 replicates) and 10-6 (just 1/3 replicates) in Namalwa Cell Line. Similar results were obtained when we characterized the IgH recombination repertoire of two CLL patients over time.Conclusions:IgH sequencing with the IT-PGM platform showed at least the same level of sensitivity as ASO RQ-PCR, without the need for patient-specific reagents. It also allows specific and detailed molecular characterization of the clonal rearrangements and could be easily incorporated into clinical laboratories for routine testing of MRD in B-cell malignancies. DisclosuresNo relevant conflicts of interest to declare.

Somatic Mutations in MLH1 and MSH2 Are a Frequent Cause of Mismatch-Repair Deficiency in Lynch Syndrome-Like Tumors

Lynch syndrome is caused by germline mutations in the mismatch repair (MMR) genes. Tumors are characterized by microsatellite instability (MSI). However, a considerable number of MSI-positive tumors have no known molecular mechanism of development. By using Sanger and ion semiconductor sequencing, 25 MSI-positive tumors were screened for somatic mutations and loss of heterozygosity in mutL homolog 1 (MLH1) and mutS homolog 2 (MSH2). In 13 of 25 tumors (8 MLH1-deficient and 5 MSH2-deficient tumors), we identified 2 somatic mutations in these genes. We conclude that 2 acquired events explain the MMR-deficiency in more than 50% of the MMR-deficient tumors without causal germline mutations or promoter methylation.

Genetic high throughput screening in Retinitis Pigmentosa based on high resolution melting (HRM) analysis

Retinitis Pigmentosa (RP) involves a group of genetically determined retinal diseases caused by a large number of mutations that result in rod photoreceptor cell death followed by gradual death of cone cells. Most cases of RP are monogenic, with more than 80 associated genes identified so far. The high number of genes and variants involved in RP, among other factors, is making the molecular characterization of RP a real challenge for many patients. Although HRM has been used for the analysis of isolated variants or single RP genes, as far as we are concerned, this is the first study that uses HRM analysis for a high-throughput screening of several RP genes. Our main goal was to test the suitability of HRM analysis as a genetic screening technique in RP, and to compare its performance with two of the most widely used NGS platforms, Illumina and PGM-Ion Torrent technologies. RP patients (n = 96) were clinically diagnosed at the Ophthalmology Department of Donostia University Hospital, Spain. We analyzed a total of 16 RP genes that meet the following inclusion criteria: 1) size: genes with transcripts of less than 4 kb; 2) number of exons: genes with up to 22 exons; and 3) prevalence: genes reported to account for, at least, 0.4% of total RP cases worldwide. For comparison purposes, RHO gene was also sequenced with Illumina (GAII; Illumina), Ion semiconductor technologies (PGM; Life Technologies) and Sanger sequencing (ABI 3130xl platform; Applied Biosystems). Detected variants were confirmed in all cases by Sanger sequencing and tested for co-segregation in the family of affected probands. We identified a total of 65 genetic variants, 15 of which (23%) were novel, in 49 out of 96 patients. Among them, 14 (4 novel) are probable disease-causing genetic variants in 7 RP genes, affecting 15 patients. Our HRM analysis-based study, proved to be a cost-effective and rapid method that provides an accurate identification of genetic RP variants. This approach is effective for medium sized (<4 kb transcript) RP genes, which constitute over 80% of the total of known RP genes.

CNVs Provide the Clue for a Cryptic t(5;11) in Acute Myeloid Leukemia

s from the Cancer Cytogenomics Microarray Consortium/CAGdb meeting, Chicago, IL, August 5e7, 2013 Massive Parallel Sequencing of Leukemia Samples Using a Custom Designed Cancer Gene Mutation Panel Fengqi Chang, Liu Liu, Erica Fang, Guangcheng Zhang, Yanchun Li, Marilyn M. Li Baylor College of Medicine, Houston, TX, USA Leukemia is the result of accumulation of somatic mutations including point mutations, copy number variations, and chromosomal abnormalities. Since multiple mutations can be present in a given leukemia genome, a technology that can detect many mutations in different genes simultaneously is desirable. Next generation sequencing (NGS) technologies allow deep sequencing of numerous mutations and multiple samples at the same time. Among NGS technologies, targeted sequencing is appealing in clinical oncology due to its shorter turnaround time, lower costs, and simpler data analysis compared to whole genome or whole exome sequencing. We introduce here a new panel specific for leukemia using multiplex PCR followed by ion semiconductor deep sequencing technologies. The leukemia panel covers 48 genes and can detect over 3,200 leukemia-associated mutations, many of which are clinically actionable mutations. Twenty-five samples were used to validate the panel on different sample types, including blood, bone marrow, FFPE and cell free DNA. The uniformity of coverage was about 87.75% for all cases. The average depth of coverage for targeted region was more than 3000X and the mean 100X coverage of targeted mutations was 97.07%. The mutation panel detected mutations in NPM1, TP53, ABL1, JAK3, IDH1, IDH2, KRAS etc in these samples with as little as 20 ng DNA and the positive rate was about 56%. The most commonly mutated genes were: TP53 (7 cases) followed by CEBPA (3 cases), NPM1 (3 cases). Overall, the leukemia panel showed improved coverage uniformity, reading length and depth compared to certain commercially available kits. The turnaround time for the test can be as short as 3 to 5 days, which fits the needs of personalized oncology. Furthermore, samples can be multiplexed using barcoding, which leads to tremendous increase of sequencing efficiency and decrease of cost while maintaining the high quality of sequencing results. CNVs Provide the Clue for a Cryptic t(5;11) in Acute Myeloid Leukemia Nicole L. Chia , Howard R. Slater , Julia M. Potter a,b NU Medical School, Australian National University, Canberra ACT 0200, Australia; ACT Pathology, The Canberra Hospital, Canberra ACT, 2606 Australia; Victorian Clinical Genetics Services, Murdoch Children’s Research Institute, Royal Children’s Hospital, Melbourne, Vic 3052, Australia Molecular karyotyping has been introduced into diagnostic pathology services for first-tier investigation of constitutional anomalies. The clinical utility of this mode of diagnostic investigation in neoplasms is currently being explored. Evidence based interpretation of copy number change requires comparison with control cohorts. Cytogenetic investigation of neoplasms frequently requires identification of apparently balanced structural rearrangements that result in critical gene fusion. High resolution microarray investigations have shown that not all balanced rearrangements are in fact balanced at the sequence level. We describe a 25 year old male with acute myelomonocytic leukemia. A del(5)(q35) was identified as the sole abnormality by conventional cytogenetic analysis. SNP microarray analysis confirmed an acquired 6.0 Mb interstitial deletion at 5q35.1q35.3 and an adjacent 350kb deletion. In addition, two acquired adjacent deletions (2.1 Mb and 850kb) were observed at chromosome 11p15.5p15.4 and a 675kb gain at 10q11.22. Subsequent subtelomere FISH identified a cryptic t(5;11)(q35;p15) with putative NUP98/NSD1 fusion. Review of the “Aussie Normal” control cohort yielded no evidence of CNVs on chromosome 5 and 11 at the putative breakpoints. However, common CNVs were observed within the deleted regions for each chromosome. These are located within repetitive DNA (LINES and SINES). A low frequency CNV gain (220kb) in the Aussie Normal cohort, harboring the ANXA8L2 gene at 10q11.2 coincided with the CNV reported in the patient here. This CNV is located within a region of segmental duplication and may represent a rearrangement due to NAHR mechanisms. This case demonstrates the potential value for the application of molecular karyotyping of neoplasms. Comparison with control cohorts provides evidence upon which pathogenicity, predisposition, and putative mechanisms can be investigated.

Deep ion sequencing of amplicon adapter ligated libraries: a novel tool in molecular diagnostics of formalin fixed and paraffin embedded tissues

Due to the advanced progress in personalised therapy concepts for non-small cell lung cancer (NSCLC), we applied the ion semiconductor sequencing (ISS) approach to molecular diagnosis of NSCLC, analysing a...

Abstract 4218: Validation of the Ion AmpliSeq™ Comprehensive Cancer Panel (CCP) using castPCR™ technologies.

Abstract Somatic mutation has been implicated in many aspects of cancer such as susceptibility, diagnosis, prognosis, drug response and tumor progress. Detection of somatic mutation is of wide interest in cancer research. The rapid advances in next generation sequencing (NGS) technologies have transformed cancer research. For example, the Ion AmpliSeq™ technology enables the selective amplification of 10s to 1000s of target sequences in a single multiplexed PCR and meshes seamlessly with the Ion semiconductor sequencing platform. The Ion Ampliseq™ Comprehensive Cancer Panel (CCP) provides ready-access to hundreds of genes, making it ideal for broad targeted re-sequencing studies. Alternative technologies are in demand to validate the NGS data orthogonally and screen hundreds or more cancer samples to evaluate mutation patterns, prevalence and frequencies in population. Here, we have demonstrated the utility of TaqMan® Mutation Detection Assays using our competitive allele specific TaqMan® PCR (castPCR™) technology in validation of Ion Torrent sequencing data. In this study, we applied the Ion Ampliseq™ Comprehensive Cancer Panel (CCP) panel to NCI-60 cell lines (MCF-7, MDA-MB-231, DU-145, PC-3, SK-MEL- 28) derived from breast, prostate, and skin cancers on Ion Torrent PGM sequencer. We confirmed previously reported mutations in these cell lines and identified the mutations that were not reported before, including missense and non-coding mutations. We then selected a subset of mutation targets from the cancer panel for castPCR validation including the genes of KRAS. EGFR, BRAF, NRAS, PIK3CA, PTEN, KIT, TP53, and more. Since limited sample quantity has been a challenging issue for most cancer researchers, especially for those who are interested in testing multi-targets by qPCR, we developed a preamplification method to enrich the targets of interest prior to running castPCR assays. We compared the Cq values of on-targets and off-targets from preamplified samples and non-preamplified samples, and the data indicates that our preamplification strategy not only provides roughly100 fold target enrichment but also maintains specificity during the preamplification process. For mutation detection, we showed that the mutation data from castPCR™ and from Ion Torrent PGM sequencer share high concordance for any given mutations. The detailed comparison and analysis will be presented and discussed. Our results demonstrate that castPCR™ technology is a valuable validation tool for next generation sequencing. The combination of Ion Torrent sequencing and castPCR validation empowers cancer researchers to understand the roles that somatic mutation plays in cancer. Citation Format: Kelly Li, Cora Woo, Mokang Mouanoutoua, Bonnie Moy, Emanuel Langit, Pius Brzoska, Xiaoqing You, Sejal Desai, David Keys, Junko Stevens, Benjamin Kong, Mark Shannon, Shiaw-Min Chen, David Ruff, Chieh-Yuan Li, David Joun, Iris Casuga, Robert Bennett. Validation of the Ion AmpliSeq™ Comprehensive Cancer Panel (CCP) using castPCR™ technologies. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4218. doi:10.1158/1538-7445.AM2013-4218

Rapid deep sequencing of patient-derived HIV with ion semiconductor technology

The development of next-generation sequencing technologies has facilitated the study of HIV drug resistance evolution. However, the high capacity and per-run cost of many sequencers is not ideal for viral sequencing unless many samples are analyzed simultaneously. Ion semiconductor sequencing has recently emerged as a flexible, lower-cost alternative with short runtime. This paper describes the use of Ion Torrent devices for deep sequencing of drug resistant HIV samples. High levels of sequencing coverage were obtained in HIV Gag and protease, allowing the detection of mutations at low frequencies.

Thomson Parabola Spectrometer for Energetic Ions Emitted from Sub-ns Laser Generated Plasmas

Laser-generated plasmas were obtained in high vacuum by irradiating micrometric thin films (Au, Au/Mylar, Mylar) with the Asterix laser at the PALS Research Infrastructure in Prague. Irradiations at the fundamental wavelength, 300 ps pulse duration, at intensities up to about 1016W/cm2, enabled ions to be accelerated in forward direction with kinetic energies of the order of 2 MeV/charge state. Protons above 2 MeV were obtained in the direction orthogonal to the target surface in selffocusing conditions. Gold ions up to about 120 MeV and 60+ charge state were detected. Ion collectors and semiconductor SiC detectors were employed in time-of-flight arrangement in order to measure the ion velocities as a function of the angle around the normal direction to the target surface. A Thomson parabola spectrometer (TPS) with a multi-channel-plate detector was used to separate the different ion contributions to the charge emission in single laser shots, and to get information on the ion charge states, energy and proton acceleration. TPS experimental spectra were compared with accurate TOSCA simulations of TPS parabolas.

ABRF 2012: Best Poster Competition

With the continuing mission of providing significant educational opportunities to the Association of Biomolecular Resource Facilities (ABRF) membership, the Education Committee (EdComm) has a proud heritage of overseeing the Waters Research Poster Competition for the annual meeting. The competition is open to all and includes scientific reports from academia, as well as cutting-edge research from core facilities and corporate R&D groups. The goal of offering the awards is to elicit the highest level of information on techniques, applications, and technologies and share knowledge pertinent to further excellence in core facility services. The process for judging the poster submissions begins with the author submitting an abstract on-line during the registration procedure. The author chooses a submission category and self-selects whether to be considered for competition. Each abstract is reviewed by at least three members of the EdComm, based on the following criteria adapted from the Annual BioMedical Research Conference for Minority Students: development of a hypothesis or statement of the problem, description of methods and controls, clarity in the presentation of results, a conclusion that summarizes the results, and impact of the results to the core facility mission. The entire committee comes to a consensus as to the semifinalists. All semifinalists must agree to give a short oral presentation during the poster review session for the EdComm and to give a 15-min talk at a regular meeting session if they are a winner. During the annual meeting, teams of EdComm members circulate through the poster session and evaluate the semifinalist presentations. The metrics used for evaluating abstracts also contain information about how to judge (rank) each of three additional aspects determined during the designated poster presentation: clarity of the poster presentation, originality and contribution to the core facility, and oral presentation. Four finalists are ultimately selected. Competition has provided the ABRF membership with excellent posters to enjoy during the annual meeting. The presentation by finalists during the annual meeting is an opportunity to obtain further information about the science behind the poster. We are fortunate to provide ABRF attendees with this opportunity and thank Waters Corporation for continuing its support of this award by providing a monetary honorarium to the winners. The EdComm proudly congratulates the winners of the ABRF 2012 Waters Best Poster Award: Alexandre Campos ProteoRed Multicenter Experiment for Long-term Quality Control Evaluation of Proteomics Core Facilities Proteomics Core Facility, Barcelona Science Park, Barcelona, Spain Toumy Guettouche An Improved Workflow for miRNA Expression Profiling Using Ion Semiconductor Sequencing Hussman Institute for Human Genomics and Sylvester Comprehensive Cancer Center, University of Miami School of Medicine, Miami, FL, USA Zhenjiu Liu Redox Protein Characterization and Quantification Using ICAT/Itraq-MS to Investigate Thiol-Based Regulatory Mechanisms Induced by Oxidative Stress in Plants Donald Danforth Plant Science Center, St. Louis, MO, USA Peter Tonner Transcriptomic Profiling of Ribosomal Protein Pseudogenes in Diverse Human Tissuesy Department of Electrical Engineering & Computer Science, University of Central Florida, Orlando, FL, USA

The efficacy of targeted next-generation sequencing for detection of clinically actionable mutations in cancer.

10598 Background: The emergence of next-generation sequencing (NGS) technologies has significantly accelerated the identification of cancer-causing mutations and the development of personalized cancer care. However, the clinical application of these technologies to detect cancer gene mutations has been extremely limited due to the long turn around time, the high cost, and large amount of input DNA required by existing NGS-based tests. Methods: We have assessed the performance of a novel NGS technology that merges multiplex PCR with ion semiconductor sequencing (AmpliSeq, Life Technologies, Inc.) in our clinical diagnostic laboratory. The test interrogates 739 common mutations in 46 cancer genes including many clinically actionable mutations concurrently. First, we studied 12 tumor samples including 4 archived FFPE, 4 blood/bone marrow, and 4 cell line samples with known mutations to evaluate the sensitivity and specificity of the test. We then studied 34 de-identified, archived FFPE tumor samples of unknown genotype to further evaluate the efficacy of the test. Results: We successfully identified all known mutations previously detected by Pyposequencing or Sanger sequencing technologies. Multiple serial dilution studies showed that the test could detect mutations at frequencies as low as 5% with 99% confidence. For the samples of unknown genotype, we detected 23 COSMIC mutations in 16 samples including HRAS, BRAF, MET, TP53 mutations in lung cancer, KRAS, PIK3CA, TP53, APC, BRAF, ERBB2 mutations in colon cancer, TP53 and KRAS mutations in breast cancer, and KIT and PDGFRA mutations in GIST. Analysis of the variant call data showed that a minimum of 100X coverage is required in order to detect mutations at 10% frequency or above; a minimum 300K final library reads are necessary in order to minimize/eliminate amplicon dropout. Conclusions: The targeted NGS test can effectively detect cancer gene mutation with input DNA as low as a few nanograms, turn around time can be as short as two days, and can significantly lower cost compared to traditional Sanger sequencing. Our experience demonstrates that this technology holds great potential for clinical use, including diagnostic and therapeutic applications.

Abstract 3674: Next generation cancer gene sequencing for clinically actionable mutations

Abstract Cancer gene mutations are important diagnostic, prognostic, and predictive biologic markers. Mutation detection in cancer tissues has been a real challenge for researchers as a cancer is often associated with multiple mutations and new mutations may occur as the disease progresses and during treatments. Essentially, all cancer tissues are a mosaic mixture of normal and abnormal cell clones. Therefore, a new technology that can target many mutations simultaneously with high sensitivity is exceedingly desirable. Targeted next generation sequencing permits deep sequencing of hundreds of mutations concurrently. We have used a novel next generation sequencing technology that merges multiplex PCR with ion semiconductor sequencing (AmpliSeq, Life Technology) to sequence a panel of clinically actionable cancer gene mutations on 12 tumor samples including 6 archived FFPE samples, 2 bone marrow samples, and 4 cell lines with known mutations, and a normal blood sample as a control. Each sample was run three times to assay for inter-run variability. The panel contains 739 mutations including clinically actionable mutations such as EGFR mutations in exons 18-21, BRAF V600E, KRAS mutations at codons 12, 13, and 61, and many more. Using this technology we successfully identified all known mutations previously detected using Pyposequencing or Sanger sequencing technologies. The most common somatic mutations were also confirmed by Competitive Allele-Specific TaqMan® PCR (castPCR, Life Technology). To determine the sensitivity of the test, we studied mutation positive FFPE samples with serial dilutions. Our results showed that the test can detect mutations at frequencies as low as 5% with 99% confidence. Analysis of mutation calls in different runs showed little inter-run variation. Other benefits of this novel technology are its fast turn around time, which can be as short as two days, and its low cost. Our experience demonstrates that this technology holds great potential clinical utility including diagnostic and therapeutic applications. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3674. doi:1538-7445.AM2012-3674

Proton emission from thin hydrogenated targets irradiated by laser pulses at 1016 W/cm2

The iodine laser at PALS Laboratory in Prague, operating at 1315 nm fundamental harmonics and at 300 ps FWHM pulse length, is employed to irradiate thin hydrogenated targets placed in vacuum at intensities on the order of 10(16) W∕cm(2). The laser-generated plasma is investigated in terms of proton and ion emission in the forward and backward directions. The time-of-flight technique, using ion collectors and semiconductor detectors, is used to measure the ion currents and the corresponding velocities and energies. Thomson parabola spectrometer is employed to separate the contribution of the ion emission from single laser shots. A particular attention is given to the proton production in terms of the maximum energy, emission yield, and angular distribution as a function of the laser energy, focal position, target thickness, and composition. Metallic and polymeric targets allow to generate protons with large energy range and different yield, depending on the laser, target composition, and target geometry properties.

Lanthanide modified semiconductor photocatalysts

Lanthanide ion modified semiconductor photocatalysts have been explored for photocatalytic degradation of organic pollutants, dye molecules, for photo-splitting of water under both ultra-violet (UV) and visible light conditions. This review provides an in-depth analysis of lanthanide ion semiconductor photocatalysts with focus on titania based photocatalysts. The emphasis is on delineating the underlying factors responsible for the enhanced activities observed by several research groups and in providing guidance to researchers in this area.

Real-Time Two-Dimensional Imaging of Potassium Ion Distribution Using an Ion Semiconductor Sensor with Charged Coupled Device Technology

Two-dimensional real-time observation of potassium ion distributions was achieved using an ion imaging device based on charge-coupled device (CCD) and metal-oxide semiconductor technologies, and an ion selective membrane. The CCD potassium ion image sensor was equipped with an array of 32 × 32 pixels (1024 pixels). It could record five frames per second with an area of 4.16 × 4.16 mm(2). Potassium ion images were produced instantly. The leaching of potassium ion from a 3.3 M KCl Ag/AgCl reference electrode was dynamically monitored in aqueous solution. The potassium ion selective membrane on the semiconductor consisted of plasticized poly(vinyl chloride) (PVC) with bis(benzo-15-crown-5). The addition of a polyhedral oligomeric silsesquioxane to the plasticized PVC membrane greatly improved adhesion of the membrane onto Si(3)N(4) of the semiconductor surface, and the potential response was stabilized. The potential response was linear from 10(-2) to 10(-5) M logarithmic concentration of potassium ion. The selectivity coefficients were K(K(+),Li(+))(pot) = 10(-2.85), K(K(+),Na(+))(pot) = 10(-2.30), K(K(+),Rb(+))(pot) =10(-1.16), and K(K(+),Cs(+))(pot) = 10(-2.05).

Differentiation of air samples with and without microbial volatile organic compounds by aspiration ion mobility spectrometry and semiconductor sensors

A chemical detector ChemPro100i containing aspiration type ion mobility spectrometry (aspiration IMS), five semiconductor sensors (MOS) and field effect transistor sensor (FET) was used for the detection and monitoring of microbial volatile organic compounds (MVOC). MVOCs were detected from the headspace of glass chambers which contained a specimen of building material in humid conditions. Results regarding particle board as a substrate for mould growth are represented. A specimen kept under sterile conditions was used as a reference. Samples were incubated for 22 days and detector responses as well as air samples introduced into Tenax adsorbent were collected for seven days during the incubation. Air samples were analyzed by thermal desorption gas chromatography mass spectrometry (TD-GC-MS) for identifying the possible MVOCs. MVOC detection was done by comparing the chromatogram peak intensities between the contaminated and the sterile samples. Detector responses were monitored before and after air sampling and data was analyzed by means of multivariate data analysis (MVDA) using SIMCA-P 10.0 software (Umetrics, Umeå, Sweden). Results showed that when incubation proceeded the different samples could be separated by both aspiration IMS combined with sensors and TD-GC-MS and that the results obtained by both methods also had a visible time trend.

Tuning transport properties of nanofluidic devices with local charge inversion.

Nanotubes can selectively conduct ions across membranes to make ionic devices with transport characteristics similar to biological ion channels and semiconductor electron devices. Depending on the surface charge profile of the nanopore, ohmic resistors, rectifiers, and diodes can be made. Here we show that a uniformly charged conical nanopore can have all these transport properties by changing the ion species and their concentrations on each side of the membrane. Moreover, the cation versus anion selectivity of the pores can be changed. We find that polyvalent cations like Ca(2+) and the trivalent cobalt sepulchrate produce localized charge inversion to change the effective pore surface charge profile from negative to positive. These effects are reversible so that the transport and selectivity characteristics of ionic devices can be tuned, much as the gate voltage tunes the properties of a semiconductor.