Semiconductor Sequencing Technology Research Articles

Multi-Gene Single Nucleotide Polymorphism Detection in Gastric Cancer Based on Ion Semiconductor Sequencing Platform.

Gastric cancer is a common heterogeneous tumor. Most patients have advanced gastric cancer at the time of diagnosis and often need chemotherapy. Although 5-fluorouracil (5-FU) is widely used for treatment, its therapeutic sensitivity and drug tolerance still need to be determined, which emphasizes the importance of individualized administration. Pharmacogenetics can guide the clinical implementation of individualized treatment. Single nucleotide polymorphisms (SNPs), as a genetic marker, contribute to the selection of appropriate chemotherapy regimens and dosages. Some SNPs are associated with folate metabolism, the therapeutic target of 5-FU. Methylenetetrahydrofolate reductase (MTHFR) rs1801131 and rs1801133, dihydrofolate reductase (DHFR) rs1650697 and rs442767, methionine synthase (MTR) rs1805087, gamma-glutamyl hydrolase (GGH) rs11545078 and solute carrier family 19 member 1 (SLC19A1) rs1051298 have been investigated in different kinds of cancers and antifolate antitumor drugs, which have potential forecasting and guiding significance for application of 5-FU. The ion torrent next-generation semiconductor sequencing technology can rapidly detect gastric cancer-related SNPs. Each time a base is extended in a DNA chain, an H+ will be released, causing local pH changes. The ionic sensor detects pH changes and converts chemical signals into digital signals, achieving sequencing by synthesis. This technique has low sample requirement, simple operation, low cost, and fast sequencing speed, which is beneficial for guiding individualized chemotherapy by SNPs.

Detection of genetic mutations in patients with breast cancer from Saudi Arabia using Ion AmpliSeq™ Cancer Hotspot Panel v.2.0.

Next-Generation Sequencing allows for quick and precise sequencing of multiple genes concurrently. Recently, this technology has been employed for the identification of novel gene mutations responsible for disease manifestation among breast cancer (BC) patients, the most common type of cancer amongst Arabian women, and the major cause of disease-associated death in women worldwide. Genomic DNA was extracted from the peripheral blood of 32 Saudi Arabian BC patients with histologically confirmed invasive BC stages I-III and IV, as well from 32 healthy Saudi Arabian women using a QIAamp® DNA Mini Kit. The isolated DNA was quantified using a Qubit™ dsDNA BR Assay Kit with a Qubit 2.0 Fluorometer. Ion semiconductor sequencing technology with an Ion S5 System and AmpliSeq™ Cancer Hotspot Panel v2 were utilized to analyze ~2,800 mutations described in the Catalogue of Somatic Mutations in Cancer from 50 oncogenes and tumor suppressor genes. Ion Reporter Software v.5.6 was used to evaluate the genomic alterations in all the samples after alignment to the hg19 human reference genome. The results showed that out of the 50 genes, 26 mutations, including 17 (65%) missense point mutations (single nucleotide variants), and 9 (35%) frameshift (insertion/deletion) mutations, were identified in 11 genes across the cohort in 61 samples (95%). Mutations were predominantly focused on two genes, PIK3CA and TP53, in the BC genomes of the sample set. PIK3CA mutation, c.1173A>G located in exon 9, was identified in 15 patients (46.9%). The TP53 mutations detected were a missense mutation (c.215C>G) in 26 patients (86.70%) and 1 frameshift mutation (c.215_216insG) in 1 patient (3.33%), located within exon 3 and 5, respectively. This study revealed specific mutation profiles for every BC patient, Thus, the results showed that Ion Torrent DNA Sequencing technology may be a possible diagnostic and prognostic method for developing personalized therapy based on the patient's individual BC genome.

Chromosomal Mosaicism in Cleavage Stage Embryos vs. Blastocyst Stage Embryos

Introduction: Chromosomal mosaicism is characterized by the presence of more than one chromosomally different cell line in an individual. Preimplantation chromosomal mosaicism is characterized by the presence of a mixture of chromosomally different cell lines in an embryo. Studies show that mosaicism for whole chromosomes (aneuploidies) in one or more cells (blastomeres) occurred in more than 75% of cleavage stage embryos, whilst 3%-24% of blastocyst stage embryos are chromosomally mosaic. Aim: The purpose of this study was to standardize and validate a Next Generation Sequencing (NGS) method for comprehensive chromosome testing for aneuploidies and to study the level of mosaicism in cleavages stage vs. blastocyst stage embryos. Methods: The validation involved a retrospective blind assessment of whole genome amplification (WGA) products from 14 cleavages stage embryo biopsies (blastomeres), 6 blastocyst stage embryo biopsies (TE), in addition to their 20 discarded blastocyst stage whole embryos. 42.8% of the cleavages stage embryos showed mosaicism, whilst results between the trophectoderm (TE) biopsies (TEB) and their whole embryos at blastocyst stage showed total concordance as no mosaicism was observed. NGS sensitivity and specificity for calling aneuploidy was found to be 100%. Conclusion: This is the first study reporting preclinical validation and accuracy assessment of the Ion semiconductor sequencing technology in studying the level of mosaicism in cleavage stage and TE biopsies blastocyst stage embryos vs. their whole embryos. The high level of mosaicism in cleavages stage embryos compared to blastocyst stage embryos does not recommend the PGT-A to be performed on cleavage stage embryos. The NGS proved to be a robust methodology in clinical application of PGT-A.

Whole-Genome Sequencing and Phylogenetic Analysis of <i>Francisella tularensis</i> Vaccine Strain 15 NIIEG

Objective of the study was to conduct whole-genome sequencing of the vaccine strain Francisella tularensis 15 NIIEG and determine, based on the results, its phylogenetic relationships and the genetic organization features.Materials and methods. Whole-genome sequencing of F. tularensis 15 NIIEG strain was performed on Ion PGM (Ion Torrent, USA) and MinIon (Oxford Nanopore Technologies, UK) platforms. Alignment of readings obtained to the whole-genome of F. tularensis subsp. holarctica LVS (CP009694, USA, 2015) was performed using the software package DNASTAR Lasergene 15.3. Hybrid assembly of reads into contigs was performed by means of Unicycler v. 0.4.4, using data obtained by semiconductor sequencing technology (Ion PGM) and nanopore sequencing (MinIon). Phylogenetic analysis was performed on the basis of single nucleotide polymorphism (SNPs) data located in the core part of F. tularensis genome. Maximum parsimony algorithm was used to construct a dendrogram using the obtained data of common SNP-matrix.Results and discussion. The close relations of F. tularensis 15 NIIEG strain with F. tularensis LVS vaccine strain used in the countries of Western Europe and North America was confirmed. Searching for common single mutations characteristic of F. tularensis 15 vaccine strains of NIIEG and LVS, permitted to find 5 unique SNPs that distinguish them from all other 228 F. tularensis strains used in the comparison. Comparative genomic analysis ofF. tularensis 15 NIIEG vaccine strain and virulent strains revealed in its structure two extensive 526 bp deletions (genes pilA and pilE) and 1480 bp (genes encoding lipoprotein). Similar deletions are also present in the genome of the F. tularensis LVS vaccine strain.

Non-toxigenic strains of Vibrio cholerae biovar El Tor, isolated in the territory of Russia: molecular-genetic peculiarities and pathogenic properties

Aim. Comparative analysis of the sequenced on our own effort genomes of non-toxigenic strains with different set of pathogenicity genes and assessment of their virulence on the model of intestinal inoculation of newborn rabbits. Materials and methods. Whole-genome DNA sequencing of 26 strains was carried out using semiconductor sequencing technology. Haemolysin and hemagglutinin-protease production was evaluated applying conventional methods. Virulence of the strains for newborn rabbits was determined through intraluminal inoculation at the dose of 107 CFU/ml. Results. On the basis of whole genome analysis of ctxA-tcpA+ and ctxA-tcpA- non-toxigenic strains, differences in composition and structure stability of mobile elements associated with pathogenicity have been identified. Significant differences in nucleotide sequences of hlyA, hapA, and rtxA genes, encoding production of additional pathogenicity factors, have also been detected. The paper provides the results of assessment of their phylogenetic relations. Experiments on animal models have confirmed the inability of non-toxigenic ctxA-tcpA+ and ctxA-tcpA- strains to cause the development of typical cholera infection. Conclusion. New data on the genome structure of different non-toxigenic strains and their phylogenetic relations have been obtained. Based on the results of inoculation of animal models with cells of non-toxigenic strains with the studied genome the inference is drawn on their inability to cause the development of typical cholera infection.

Cytogenetic and molecular characterization of an oligoasthenozoospermia male carrier of an unbalanced Y;22 translocation

Rationale:Y;autosome translocations are associated with male infertility and azoospermia. Some carriers with a Y:22 translocation can produce offspring and transmit the translocation through generations without phenotypic repercussion. Hence, the clinical features of carriers with certain Y chromosome abnormalities remain uncertain.Patient concerns:An apparently healthy 33-year-old man, 175 cm tall and weighing 60 kg had a 6-month history of primary infertility.Diagnoses:The patient was diagnosed with oligoasthenozoospermia. A series of examinations have been performed to evaluate possible genetic causes of this diagnosis. Several methods included semen analysis, hormone measurements, cytogenetic analysis, and high-throughput multiplex ligation-dependent probe amplification semiconductor sequencing.Interventions:The patient underwent detailed genetic counseling. Cytogenetic analysis was advised for his father. Preimplantation genetic diagnosis was performed to improve potential pregnancy success rate.Outcomes:Semen analysis revealed oligoasthenozoospermia. Hormone levels were within the normal limits. The karyotype of the patient and his father was 45,X,der(Y;22). Sequencing results indicated the presence of the sex-determining region on the Y chromosome gene. Y-chromosome microdeletion detection showed the presence of AZF (azoospermic factor)a, AZFb, and AZFc regions, but deletion of b2/b3 and duplication of b3/b4 regions.Lessons:A clinical karyotype report involving a Y chromosome abnormality should consider the results of semen analysis, which helps to identify the chromosomal breakpoint. Semiconductor sequencing technology was useful for clarifying AZF gene microdeletions.

Validation and Clinical Impact of the Oncomine Myeloid Targeted DNA and RNA Ion Semiconductor Sequencing Assay

Introduction: The expanding diagnostic and clinical significance of genes associated with myeloid malignancies necessitates higher throughput testing. Targeted next-generation sequencing (NGS) of DNA and RNA may be an ideal approach to capture these variants and eventually streamline, or replace, conventional cytogenetic testing. We present our clinical validation of the OncomineTM Myeloid NGS assay and early experience regarding the clinical impact in patients with suspected or known myeloid malignancies.Methods: OncomineTM Myeloid (Thermo Fisher Scientific) panel (OMP) validation was conducted at Kingston Health Sciences Centre (KHSC) on anonymized and commercial samples according to IQMH and ACMG standards for NGS (PMID: 23887774). As of April 2018 the test has been formally validated and offered at KHSC. Prior to this, patients were consented at the time of blood (PB) or bone marrow (BM) collection for research-based testing. BM and PB samples were collected, gDNA and RNA extracted, and libraries prepared using the Ion ChefTM System. Sequencing was completed using the OMP on Ion PGM (research phase) and S5XL (clinical) sequencers. The OMP uses amplicon-based semiconductor sequencing technology to cover 40 DNA genes recurrently mutated in myeloid malignancies (23 hotspots and 17 full coding), 29 RNA fusion driver genes and 10 gene expression targets. Sequencing data were analyzed using Torrent Suite, Torrent Variant Caller, Ion Reporter (v.5.2-5.6) and Alamut software. The OMP includes a Torrent Suite FLT3-ITD “plug-in” and Ion Reporter “Oncomine filter”. Orthogonal clinical assays were performed where necessary for additional confirmation. Variants were interpreted based on a 4-tier classification system developed by Li et al (PMID: 27993330). Variants in the top 3 tiers were reported after assessment by at least one clinician and one certified medical geneticist. Actionability was assessed in the context of electronic diagnostic and clinical information and molecular board review.Results: Validation involved 24 paired DNA-RNA samples previously run at KHSC on an early access OMP version and also at an external site (kind exchange by Dr. N. Carson, Saint John, NB), along with SeraCareTM DNA and RNA controls. Validation run metrics were as follows: DNA means (mapped reads 1306216, read length 226bp, uniformity 98%, depth 2518), RNA means (mapped reads 260350, read length 114bp). Complete concordance was found for RNA variants while the DNA filter chain was only modified to better capture a JAK2 exon 12 variant in a SeraCare control. The minimum depth of coverage was subsequently set to 400X for somatic DNA variants and sensitivity set at 5% variant allele fraction (VAF) for clinical reporting. To date, we have performed sequencing for 38 samples from 35 unique patients (avg age = 64.5y). Of these, 12/35 (34%) involved suspected or known myelodysplastic syndromes (MDS), 12/35 (34%) with acute myeloid leukemia (AML), 4/35 (12%) with query myeloproliferative neoplasms (MPNs), 2/35 (5%) with MDS/MPN, and 5/35 (14%) other (2 B-ALL, 1 aplastic, 1 hairy cell + ?MDS, 1 ?CHIP). OMP detected 56 variants (in 80% of cases), including 43% tier 1, 48% tier 2, and 9% tier 3 variants. The most frequently mutated genes included TET2 (21%), SRSF2 (14%), SF3B1 (9%), TP53 (5%), and RUNX1 (5%). The average VAF was 36% for all samples, with an average of 1.5 variants per sample. Of detected variants, 73% were potentially actionable. Specifically, 51% of actionable variants facilitated or clarified diagnosis (38% of total variants), 32% (23%) affected prognosis, and 17% (13%) had the potential to affect treatment decisions. Among 10 patients with suspected (but subthreshold for) MDS or MPN, clonality was detected in 5, while 5 had no variants, prompting further investigation of reversible cytopenia or cytosis. We are rapidly accruing patients and expect to have OMP profiles for at least 100 patients at the time of the ASH 2018 meeting.Conclusions: Oncomine Myeloid DNA and RNA molecular profiling has found greatest clinical use at our centre in the realm of MDS and AML. In the majority of patients, OMP permits the identification of actionable variants that may aid clinicians in achieving or clarifying diagnoses, inform follow-up and prognosis, and guide treatment decisions. The OMP represents a promising strategy to address the challenge of expanding, important molecular features of myeloid malignancies. DisclosuresNo relevant conflicts of interest to declare.

Fecal microbiota in the female prairie vole (Microtus ochrogaster).

We examined the fecal microbiota of female prairie voles. This species is socially and, likely, sexually monogamous, and thus serves as a valuable model in which to examine the interaction between the microbiota-gut-brain axis and social behavior. At present, little is known about the gastrointestinal microbiota of prairie voles; therefore, we performed a first characterization of the fecal microbiota using 16S rRNA gene amplicon sequencing. Semiconductor sequencing technology on an Ion Torrent PGM platform was used to assess the composition of fecal microbiotas from twelve female prairie voles. Following quality filtering, 1,017,756 sequencing reads were classified from phylum to genus level. At the phylum level, Firmicutes, Bacteroidetes, and Saccharibacteria were the predominant taxa, while the Bacteriodales, Erysipelotrichaceae, Ruminococcaceae, and Lachnospiraceae contributed the most dominant microbial groups and genera. Microbial community membership was most similar between vole sibling pairs, but consideration of taxon abundances weakened these associations. The interdependence of host factors such as genetics and behavior with the gastrointestinal microbiota is likely to be particularly pronounced in prairie voles. Our pilot characterization of the prairie vole intestinal microbiota revealed a microbial community composition remarkably consistent with the monogastric alimentary system of these rodents and their diet rich in complex plant carbohydrates. The highly social nature of these animals poses specific challenges to microbiome analyses that nonetheless are valuable for advancing research on the microbiota-gut-brain-behavior axis. Our study provides an important basis for future microbiome research in this emerging model organism for studying social behavior.

Clinical Experience of Non-Invasive Prenatal Chromosomal Aneuploidy Testing in 190,277 Patient Samples.

To detect trisomy 21, 18, and 13 in 190,277 clinical samples from the medical diagnostic laboratories of ten hospitals. The study assessed the clinical performance of non-invasive prenatal testing (NIPT) in detecting trisomy 21, 18, and 13 in 190,277 clinical samples using semiconductor sequencing technology. NIPT participants were at a mean gestation of 17.79 weeks (range, 9-36) and age of 31.12 years (range, 18-46) at the time of testing on average. There were 1,543 (0.81%) positive cases, including 1050 for trisomy 21, 316 for trisomy 18, and 177 for trisomy, 13. The overall sensitivity and specificity for detecting trisomy 21, 18 and 13 combined were 99.61% and 99.91% respectively, and the overall positive predictive value and negative predictive value (PPV and NPV) were 89.74% and 99.99%, respectively. This was the first large clinical study for semiconductor sequencing technologies in NIPT application. Our findings indicate that NIPT has the potential to replace serum biochemistry screening and could be performed at the early gestational age of 9~12 weeks.

Extensive next-generation sequencing analysis in chronic lymphocytic leukemia at diagnosis: clinical and biological correlations.

BackgroundIn chronic lymphocytic leukemia (CLL), next-generation sequencing (NGS) analysis represents a sensitive, reproducible, and resource-efficient technique for routine screening of gene mutations.MethodsWe performed an extensive biologic characterization of newly diagnosed CLL, including NGS analysis of 20 genes frequently mutated in CLL and karyotype analysis to assess whether NGS and karyotype results could be of clinical relevance in the refinement of prognosis and assessment of risk of progression. The genomic DNA from peripheral blood samples of 200 consecutive CLL patients was analyzed using Ion Torrent Personal Genome Machine, a NGS platform that uses semiconductor sequencing technology. Karyotype analysis was performed using efficient mitogens.ResultsMutations were detected in 42.0 % of cases with 42.8 % of mutated patients presenting 2 or more mutations. The presence of mutations by NGS was associated with unmutated IGHV gene (p = 0.009), CD38 positivity (p = 0.010), risk stratification by fluorescence in situ hybridization (FISH) (p < 0.001), and the complex karyotype (p = 0.003). A high risk as assessed by FISH analysis was associated with mutations affecting TP53 (p = 0.012), BIRC3 (p = 0.003), and FBXW7 (p = 0.003) while the complex karyotype was significantly associated with TP53, ATM, and MYD88 mutations (p = 0.003, 0.018, and 0.001, respectively). By multivariate analysis, the multi-hit profile (≥2 mutations by NGS) was independently associated with a shorter time to first treatment (p = 0.004) along with TP53 disruption (p = 0.040), IGHV unmutated status (p < 0.001), and advanced stage (p < 0.001). Advanced stage (p = 0.010), TP53 disruption (p < 0.001), IGHV unmutated status (p = 0.020), and the complex karyotype (p = 0.007) were independently associated with a shorter overall survival.ConclusionsAt diagnosis, an extensive biologic characterization including NGS and karyotype analyses using novel mitogens may offer new perspectives for a better refinement of risk stratification that could be of help in the clinical management of CLL patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-016-0320-z) contains supplementary material, which is available to authorized users.

Mutations in Driver Genes and Changes in Clonal Dynamics Are Associated with Shorter Time to Treatment in MBL Cases

Mutations in Driver Genes and Changes in Clonal Dynamics Are Associated with Shorter Time to Treatment in MBL Cases

Application of next-generation semiconductor sequencing technologies in genetic diagnosis of inherited cardiomyopathies

Inherited cardiomyopathy is the most common hereditary cardiac disease. It also causes a significant proportion of sudden cardiac deaths in young adults and athletes. So far, approximately one hundred genes have been reported to be involved in cardiomyopathies through different mechanisms. Therefore, the identification of the genetic basis and disease mechanisms of cardiomyopathies are important for establishing a clinical diagnosis and genetic testing. Next-generation semiconductor sequencing (NGSS) technology platform is a high-throughput sequencer capable of analyzing clinically derived genomes with high productivity, sensitivity and specificity. It was launched in 2010 by Life Technologies of USA, and it is based on a high density semiconductor chip, which was covered with tens of thousands of wells. NGSS has been successfully used in candidate gene mutation screening to identify hereditary disease. In this review, we summarize these genetic variations, challenge and application of NGSS in inherited cardiomyopathy, and its value in disease diagnosis, prevention and treatment.

Less is more: extreme genome complexity reduction with ddRAD using Ion Torrent semiconductor technology.

Massively parallel sequencing a small proportion of the whole genome at high coverage enables answering a wide range of questions from molecular evolution and evolutionary biology to animal and plant breeding and forensics. In this study, we describe the development of restriction-site associated DNA (RAD) sequencing approach for Ion Torrent PGM platform. Our protocol results in extreme genome complexity reduction using two rare-cutting restriction enzymes and strict size selection of the library allowing sequencing of a relatively small number of genomic fragments with high sequencing depth. We applied this approach to a common freshwater fish species, the Eurasian perch (Perca fluviatilis L.), and generated over 2.2 MB of novel sequence data consisting of ~17,000 contigs, identified 1259 single nucleotide polymorphisms (SNPs). We also estimated genetic differentiation between the DNA pools from freshwater (Lake Peipus) and brackish water (the Baltic Sea) populations and identified SNPs with the strongest signal of differentiation that could be used for robust individual assignment in the future. This work represents an important step towards developing genomic resources and genetic tools for the Eurasian perch. We expect that our ddRAD sequencing protocol for semiconductor sequencing technology will be useful alternative for currently available RAD protocols.

Small-cell lung cancer detection in never-smokers: clinical characteristics and multigene mutation profiling using targeted next-generation sequencing

BackgroundOnce regarded as a smoker's disease, small-cell lung cancer (SCLC) has been occasionally detected in never-smokers as smoking rates decrease worldwide. We investigated the clinical and genetic characteristics of SCLC in never-smokers. Patients and methodsPatients diagnosed with SCLC were grouped into smokers and never-smokers. The clinical outcomes of the two groups were compared. For SCLC in never-smokers, somatic mutation profiling was carried out using the AmpliSeq™ Cancer Hotspot Panel v2 and semiconductor sequencing technology. Epidermal growth factor receptor (EGFR) mutation was confirmed by PNAClamp™. ResultsIn total, 391 SCLC patients treated over a 5-year period were analyzed. Fifty patients (13%) were never-smokers. The median overall survival was 18.2 months in never-smokers and 13.1 months in smokers (P = 0.054). Never-smoking history was independently a good prognostic factor [hazard ratio = 0.645, 95% confidence interval (CI) 0.456–0.914], as were limited disease (HR = 0.372, 95% CI 0.294–0.471), and lower age (HR = 0.709, 95% CI 0.566–0.888). The objective response rates to first-line etoposide/cisplatin therapy were similar between never-smokers and smokers (75% versus 81%). Of 28 genetically evaluable never-smokers, EGFR mutations were detected in four cases (two L858R, one deletion in exon 19, and one G719A). Other mutations were in TP53 (n = 26), RB1 (n = 7), PTEN (n = 5), MET (n = 4), and SMAD4 (n = 3). ConclusionsNever-smokers with SCLC are increasingly prevalent and have a better prognosis than smokers with SCLC in Korea. Our study warrants further investigation in this group.

Detection of very long antisense transcripts by whole transcriptome RNA-Seq analysis of Listeria monocytogenes by semiconductor sequencing technology.

The Gram-positive bacterium Listeria monocytogenes is the causative agent of listeriosis, a severe food-borne infection characterised by abortion, septicaemia, or meningoencephalitis. L. monocytogenes causes outbreaks of febrile gastroenteritis and accounts for community-acquired bacterial meningitis in humans. Listeriosis has one of the highest mortality rates (up to 30%) of all food-borne infections. This human pathogenic bacterium is an important model organism for biomedical research to investigate cell-mediated immunity. L. monocytogenes is also one of the best characterised bacterial systems for the molecular analysis of intracellular parasitism. Recently several transcriptomic studies have also made the ubiquitous distributed bacterium as a model to understand mechanisms of gene regulation from the environment to the infected host on the level of mRNA and non-coding RNAs (ncRNAs). We have used semiconductor sequencing technology for RNA-seq to investigate the repertoire of listerial ncRNAs under extra- and intracellular growth conditions. Furthermore, we applied a new bioinformatic analysis pipeline for detection, comparative genomics and structural conservation to identify ncRNAs. With this work, in total, 741 ncRNA locations of potential ncRNA candidates are now known for L. monocytogenes, of which 611 ncRNA candidates were identified by RNA-seq. 441 transcribed ncRNAs have never been described before. Among these, we identified novel long non-coding antisense RNAs with a length of up to 5,400 nt e.g. opposite to genes coding for internalins, methylases or a high-affinity potassium uptake system, namely the kdpABC operon, which were confirmed by qRT-PCR analysis. RNA-seq, comparative genomics and structural conservation of L. monocytogenes ncRNAs illustrate that this human pathogen uses a large number and repertoire of ncRNA including novel long antisense RNAs, which could be important for intracellular survival within the infected eukaryotic host.

Classification and characterization of a laboratory chicken rotavirus strain carrying G7P[35] neutralization antigens on the genotype 4 backbone gene configuration

The laboratory rotavirus strain, BRS/115, has been used for more than two decades to monitor rotaviruses in specific pathogen free flocks of laying hens. However, the virus strain has not been characterized in detail. Therefore we aimed at the description of molecular features of BRS115 by using random primed reverse transcription-PCR of the genomic RNA followed by massive parallel sequencing using the semiconductor sequencing technology. Over 64,000 trimmed reads mapped to reference sequences obtained from GenBank. The strain classified into the species Rotavirus A and genotyped G7-P[35]-I4-R4-C4-M4-A16-N4-T4-E11-H4 according to guidelines of the Rotavirus Classification Working Group. Phylogenetic analysis identified shared features with chicken, turkey and pigeon origin rotaviruses. This study demonstrates the robustness of next generation sequencing in the characterization of reference virus materials used in specialized laboratories.

Radiation Resistance of Sequencing Chips for in situ Life Detection

Life beyond Earth may be based on RNA or DNA if such life is related to life on Earth through shared ancestry due to meteoritic exchange, such as may be the case for Mars, or if delivery of similar building blocks to habitable environments has biased the evolution of life toward utilizing nucleic acids. In this case, in situ sequencing is a powerful approach to identify and characterize such life without the limitations or expense of returning samples to Earth, and can monitor forward contamination. A new semiconductor sequencing technology based on sensing hydrogen ions released during nucleotide incorporation can enable massively parallel sequencing in a small, robust, optics-free CMOS chip format. We demonstrate that these sequencing chips survive several analogues of space radiation at doses consistent with a 2-year Mars mission, including protons with solar particle event-distributed energy levels and 1 GeV oxygen and iron ions. We find no measurable impact of irradiation at 1 and 5 Gy doses on sequencing quality nor on low-level hardware characteristics. Further testing is required to study the impacts of soft errors as well as to characterize performance under neutron and gamma irradiation and at higher doses, which would be expected during operation in environments with significant trapped energetic particles such as during a mission to Europa. Our results support future efforts to use in situ sequencing to test theories of panspermia and/or whether life has a common chemical basis.

Feasibility Study of Semiconductor Sequencing for Noninvasive Prenatal Detection of Fetal Aneuploidy

Noninvasive prenatal detection of common fetal aneuploidies with cell-free DNA from maternal plasma has been achieved with high-throughput next-generation sequencing platforms. Turnaround times for previously tested platforms are still unsatisfactory for clinical applications, however, because of the time spent on sequencing. The development of semiconductor sequencing technology has provided a way to shorten overall run times. We studied the feasibility of using semiconductor sequencing technology for the noninvasive detection of fetal aneuploidy. Maternal plasma DNA from 13 pregnant women, corresponding to 4 euploid, 6 trisomy 21 (T21), 2 trisomy 18 (T18), and 1 trisomy 13 (T13) pregnancies, were sequenced on the Ion Torrent Personal Genome Machine sequencer platform with 318 chips. The data were analyzed with the T statistic method after correcting for GC bias, and the T value was calculated as an indicator of fetal aneuploidy. We obtained a mean of 3 524 401 high-quality reads per sample, with an efficiency rate of 77.9%. All of the T21, T13, and T18 fetuses could be clearly distinguished from euploid fetuses, and the time spent on library preparation and sequencing was 24 h. Semiconductor sequencing represents a suitable technology for the noninvasive prenatal detection of fetal aneuploidy. With this platform, sequencing times can be substantially reduced; however, a further larger-scale study is needed to determine the imprecision of noninvasive fetal aneuploidy detection with this system.

Abstract 4212: Deep sequencing analysis of the ALK kinase domain identifies resistant mutations in relapse patients following crizotinib treatment.

Abstract ALK resistance mutations conferring resistance to Xalkori vary widely within the tyrosine kinase domain. So far, nine different acquired resistant mutations have been identified in the clinic, including 1151Tins, L1152R, C1156Y, F1174C, L1196M, G1202R, D1203N, S1206Y, and, G1296A. We developed a deep sequencing method using semiconductor sequencing technology to quickly detect resistance mutations within the ALK kinase domain in tumor biopsies. By bar-coding, we were able to sequence multiple samples in a single run. We have also developed an algorithm to determine assay noisy level and call mutations automatically. The assay sensitivity to call close to 1% mutated allele demonstrated by a titration experiment with a cell line harboring a know mutation. Genomic DNA analysis from relapsed patients revealed several known mutations. Our assay demonstrates robust and sensitive detection of ALK kinase mutations in NSCLC tumor samples and aids in the elucidation of resistance mechanisms pertinent to the clinical setting. Citation Format: Stephen Huang. Deep sequencing analysis of the ALK kinase domain identifies resistant mutations in relapse patients following crizotinib treatment. [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 4212. doi:10.1158/1538-7445.AM2013-4212

Multiplexed deep sequencing analysis of ALK kinase domain identifies resistance mutations in relapsed patients following crizotinib treatment

The recently approved ALK kinase inhibitor crizotinib has demonstrated successful treatment of metastatic and late stage ALK fusion positive non-small cell lung cancer (NSCLC). However, the median duration of clinical benefit is ~10–11months due to the emergence of multiple and simultaneous resistance mechanisms in these tumors. Mutations in the ALK kinase domain confer resistance to crizotinib in about one-third of these patients. We developed a multiplex deep sequencing method using semiconductor sequencing technology to quickly detect resistance mutations within the ALK kinase domain from tumor biopsies. By applying a base-pair specific error-weighted mutation calling algorithm (BASCA) that we developed for this assay, genomic DNA analysis from thirteen relapsed patients revealed three known crizotinib resistance mutations, C1156Y, L1196M and G1269A. Our assay demonstrates robust and sensitive detection of ALK kinase mutations in NSCLC tumor samples and aids in the elucidation of resistance mechanisms pertinent to the clinical setting.