Ion Torrent Platform Research Articles

Occurrence of paternal HLA-A/B and maternal HLA-B/DRB1 recombinants in one family

Using high resolution HLA typing, crossing over (CO) between HLA-A/B, B/DRB1, or DRB1/DPB1 loci can be identified among siblings from families with both parents or with large sibship. Locating the breaking points along the C I and II genomic regions can be estimated via SNP genotyping of the MHC region. We report the rare occurrence of two recombinant children in a single family and the application of SNP genotyping to pinpoint the CO in each. Blood specimens of 6 siblings from a HSCT family (F) were HLA-A/B/C/DRB1/DQB1/DPB1 typed by LABType (OLI), SBT (SeCore, Life Tech) and NGS (NXType on the Ion Torrent platform, OLI) methods. SNP genotyping for the six siblings was performed using the OMNIExpress-24 v1.1 to query over 713 K markers throughout the genome. Within the MHC there were 3754 SNPs covering the region between HLA-A position 29913067 and DQB1 position 32628428 a distance of 2.7 Mb. 100% concordant results were obtained from all three methods (Table 1). The 4 parental haplotypes segregated appropriately among siblings #3–6. This fact helps to determine sibling #1 and sibling #2 are paternal HLA-A & B and maternal HLA-B & DRB1 recombinants, respectively. The derivation of SNP haplotypes for the 6 siblings revealed the approximate breaking points where CO most likely have occurred for siblings 1 and 2. The actual breaking points were identified to have occurred within 700 bp in two respective regions, one centromerically to HLA-A for sibling 1 and the other centromerically to HLA-B for sibling 2. Precise break points will be discussed when analysis has been completed. Conclusion Two HLA recombinant children in family F have been identified. The probability of having the first two older children born as recombinants is (0.01) 2 (0.99) 4 when the birth order is specified. If birth order is not specified, the probability that two out of six children would be recombinants becomes N(N - 1)/2 x (0.01) 2 (0.99) 4 . N denotes number of children in any family. In our case, the probability is 15(0.01) 2 (0.99) 4 . Download full-size image

Contrasting transcriptome response to thermal stress in two key zooplankton species, Calanus finmarchicus and C. glacialis

MEPS Marine Ecology Progress Series Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections MEPS 534:79-93 (2015) - DOI: https://doi.org/10.3354/meps11398 Contrasting transcriptome response to thermal stress in two key zooplankton species, Calanus finmarchicus and C. glacialis Irina Smolina1,*, Spyros Kollias1, Eva F. Møller2, Penelope Lindeque3, Arvind Y. M. Sundaram4, Jorge M. O. Fernandes1, Galice Hoarau1 1Faculty of Biosciences and Aquaculture, University of Nordland, 8049 Bodø, Norway 2Department of Bioscience, Aarhus University, Frederiksborgvej 399, 4000 Roskilde, Denmark 3Plymouth Marine Laboratory, Prospect Place, West Hoe, Plymouth PL1 3DH, UK 4Norwegian Sequencing Centre, Department of Medical Genetics, Oslo University Hospital, Kirkeveien 166, 0407 Oslo, Norway *Corresponding author: ism@uin.no ABSTRACT: Climate change has already led to the range expansion of warm-water plankton assemblages in the northeast Atlantic and the corresponding range contraction of colder-water species. The temperate copepod Calanus finmarchicus is predicted to shift farther northward into polar waters traditionally dominated by the arctic copepod C. glacialis. To identify temperature-mediated changes in gene expression that may be critical for the thermal acclimation and resilience of the 2 Calanus spp., we conducted a whole transcriptome profiling using RNA-seq on an Ion Torrent platform. Transcriptome responses of C. finmarchicus and C. glacialis from Disko Bay, west Greenland, were investigated under realistic thermal stresses (at +5, +10 and +15°C) for 4 h and 6 d. C. finmarchicus showed a strong response to temperature and duration of stress, involving up-regulation of genes related to protein folding, transcription, translation and metabolism. In sharp contrast, C. glacialis displayed only low-magnitude changes in gene expression in response to temperature and duration of stress. Differences in the thermal responses of the 2 species, particularly the lack of thermal stress response in C. glacialis, are in line with laboratory and field observations and suggest a vulnerability of C. glacialis to climate change. KEY WORDS: RNA-seq · Copepods · Temperature stress response · Heat shock protein · Climate change Full text in pdf format Supplement 1 Supplement 2 PreviousNextCite this article as: Smolina I, Kollias S, Møller EF, Lindeque P, Sundaram AYM, Fernandes JMO, Hoarau G (2015) Contrasting transcriptome response to thermal stress in two key zooplankton species, Calanus finmarchicus and C. glacialis. Mar Ecol Prog Ser 534:79-93. https://doi.org/10.3354/meps11398 Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in MEPS Vol. 534. Online publication date: August 27, 2015 Print ISSN: 0171-8630; Online ISSN: 1616-1599 Copyright © 2015 Inter-Research.

Factors affecting the success of next-generation sequencing in cytology specimens.

The use of cytology specimens for next-generation sequencing (NGS) is particularly challenging because of the unconventional substrate of smears and the often limited sample volume. An analysis of factors affecting NGS testing in cytologic samples may help to increase the frequency of successful testing. This study reviewed variables associated with all in-house cytology cases (n = 207) that were analyzed by NGS with the Ion Torrent platform during a 10-month interval. A statistical analysis was performed to measure the effects of the DNA input threshold, specimen preparation, slide type, tumor fraction, DNA yield, and cytopathologist bias. One hundred sixty-four of 207 cases (79%) were successfully sequenced by NGS; 43 (21%) failed because of either a low DNA yield or a template/library preparation failure. The median estimated tumor fraction and DNA concentration for the successfully sequenced cases were 70% and 2.5 ng/μL, respectively, whereas they were 60% and 0.2 ng/μL, respectively, for NGS failures. Cell block sections were tested in 91 cases, and smears were used in 116 cases. NGS success positively correlated with the DNA yield but not the tumor fraction. Cell block preparations showed a higher success rate than smears. Frosted-tip slides yielded significantly more DNA than fully frosted slides. Lowering the input DNA concentration below the manufacturer's recommended threshold of 10 ng (>0.85 ng/μL) resulted in a marked increase in the NGS success rate from 58.6% to 89.8%. The failure of NGS with cytology samples is usually a result of suboptimal DNA due to multiple pre-analytical factors. Knowledge of these factors will allow better selection of cytology material for mutational analysis.

Small RNA deep sequencing discriminates subsets of extracellular vesicles released by melanoma cells – Evidence of unique microRNA cargos

Melanoma cells release different types of extracellular vesicles (EVs) into the extracellular milieu that are involved with communication and signaling in the tumor microenvironment. Subsets of EVs include exosomes, microvesicles, and apoptotic bodies that carry protein and genetic (RNA) cargos. To define the contribution of the RNA cargo of melanoma cell derived EVs we performed small RNA sequencing to identify different small RNAs in the EV subsets. Using validated centrifugation protocols, we separated these EV subsets released by the melanoma cell line MML-1, and performed RNA sequencing with the Ion Torrent platform. Various, but different, non-coding RNAs were detected in the EV subsets, including microRNA, mitochondrial associated tRNA, small nucleolar RNA, small nuclear RNA, Ro associated Y-RNA, vault RNA and Y-RNA. We identified in total 1041 miRNAs in cells and EV subsets. Hierarchical clustering showed enrichment of specific miRNAs in exosomes, including hsa-miR-214-3p, hsa-miR-199a-3p and hsa-miR-155-5p, all being associated with melanoma progression. Comparison of exosomal miRNAs with miRNAs in clinical melanoma samples indicate that multiple miRNAs in exosomes also are expressed specifically in melanoma tissues, but not in benign naevi. This study shows for the first time the presence of distinct small RNAs in subsets of EVs released by melanoma cells, with significant similarities to clinical melanoma tissue, and provides unique insights into the contribution of EV associated extracellular RNA in cancer.

Ion torrent next-generation sequencing reveals the complete mitochondrial genome of endangered mahseer Tor khudree (Sykes, 1839)

The complete mitochondrial genome of an endangered mahseer (Deccan mahseer), Tor khudree was sequenced using Ion torrent platform for the first time. The genome sequence was 16 573 bp in size, and consists of 13 protein coding genes, 22 tRNAs, 2 rRNA genes and 1 control region. The gene organization and its order were similar to other vertebrates. The overall base composition was A: 31.9%, G: 15.6%, C: 27.68%, T: 24.76%, A + T content 56.6% and the G + C content 43.32%. The phylogenetic tree constructed using a maximum likelihood model showed sister relationship between T. khudree and Tor tambroides.

Detecting nuisance species using NGST: Methodology shortcomings and possible application in ballast water monitoring

Detecting the presence of potential invasive species in ballast water is a priority for preventing their spread into new environments. Next generation sequencing technologies are being increasingly used for exploring and assessing biodiversity from environmental samples. Here we apply high throughput sequencing from DNA extracted from ballast water (BW) samples employing two different platforms, Ion Torrent and 454, and compare the putative species catalogues from the resulting Operational Taxonomic Units (OTU). Water samples were taken from the RV Polastern ballast tank in five different days between the second and the twentieth navigation day. Pronounced decrease of oxygen concentration and increase of temperature occurred in the BW during this time, coincident with a progressively higher proportion of unassigned OTU and short reads indicating DNA degradation. Discrepancy between platforms for species catalogues was consistent with previously published bias in AT-rich sequences for Ion Torrent platform. Some putative species detected from the two platforms increased in frequency during the Polarstern travel, which suggests they were alive and therefore tolerant to adverse conditions. OTU assigned to the highly invasive red alga Polysiphonia have been detected at low but increasing frequency from the two platforms. Although in this moment NGST could not replace current methods of sampling, sorting and individual taxonomic identification of BW biota, it has potential as an exploratory methodology especially for detecting scarce species.

Uncovering the molecular pathogenesis of congenital hyperinsulinism by panel gene sequencing in 32 Chinese patients.

Congenital hyperinsulinism (CHI) has been mostly associated with mutations in seven major genes. We retrospectively reviewed a cohort of 32 patients with CHI. Extensive mutational analysis (ABCC8,KCNJ11,GCK,GLUD1,HADH,HNF4A, and UCP2) was performed on Ion torrent platform, which could analyze hundreds of genes simultaneously with ultrahigh-multiplex PCR using up to 6144 primer pairs in a single primer pool and address time-sensitive samples with single-day assays, from samples to annotated variants, to identify the genetic etiology of this disease. Thirty-seven sequence changes were identified, including in ABCC8/KCNJ11 (n=25, 65.7%), GCK (n=2), HNF4A (n=3), GLUD1 (n=2), HADH (n=4), and UCP2 (n=1); these mutations included 14 disease-causing mutations, eight rare SNPs, 14 common SNPs, and one novel mutation. Mutations were identified in 21 of 32 patients (65.6%). Among the patients with an identified mutation, 14 had mutations in ABCC8, one of which was combined with a GLUD1 mutation. Four patients had mutations in KCNJ11, 1 had a GCK mutation, 1 had a mutation in HADH, and two had a mutation in HNF4A. Among the 32 patients, the age at the onset of hyperinsulinemia ranged from the neonatal period to 1year of age; five patients underwent a pancreatectomy due to intractable hyperinsulinemia. This study describes novel and previously identified mutations in patients with CHI. The spectrum of mutations in CHI patients represents an important tool for the diagnosis and prognosis of CHI patients in the Chinese population as well as for the genetic counseling of CHI families.

Targeted ultradeep next-generation sequencing as a method for KIT D816V mutation analysis in mastocytosis.

Next-generation sequencing (NGS) is becoming increasingly used for diagnostic mutation analysis in myeloid neoplasms and may also represent a feasible technique in mastocytosis. However, detection of the KIT D816V mutation requires a highly sensitive method in most patients due to the typically low mutation levels. In this study, we established an NGS-based KIT mutation analysis and analyzed the sensitivity of D816V detection using the Ion Torrent platform. Eighty-two individual NGS analyses were included in the study. All samples were also analyzed using highly sensitive KIT D816V mutation-specific qPCR. Measurements of the background level in D816V-negative samples supported a cutoff for positivity of 0.2% in three different NGS panels. Clinical samples from patients with SM that tested positive using qPCR with a D816V allele burden >0.2% also tested positive using NGS. Samples that tested positive using qPCR with an allele burden <0.2% tested negative using NGS. We thereby demonstrate that caution should be taken when using the potentially very sensitive NGS technique for KIT D816V mutation analysis in mastocytosis, as many patients with SM have D816V mutation levels below the detection limit of NGS. A dedicated and highly sensitive KIT D816V mutation analysis therefore remains important in mastocytosis diagnostics.

Inhibition of the endosymbiont "Candidatus Midichloria mitochondrii" during 16S rRNA gene profiling reveals potential pathogens in Ixodes ticks from Australia.

BackgroundThe Australian paralysis tick (Ixodes holocyclus) is of significant medical and veterinary importance as a cause of dermatological and neurological disease, yet there is currently limited information about the bacterial communities harboured by these ticks and the risk of infectious disease transmission to humans and domestic animals. Ongoing controversy about the presence of Borrelia burgdorferi sensu lato (the aetiological agent of Lyme disease) in Australia increases the need to accurately identify and characterise bacteria harboured by I. holocyclus ticks.MethodsUniversal PCR primers were used to amplify the V1-2 hyper-variable region of bacterial 16S rRNA genes present in DNA samples from I. holocyclus and I. ricinus ticks, collected in Australia and Germany respectively. The 16S amplicons were purified, sequenced on the Ion Torrent platform, and analysed in USEARCH, QIIME, and BLAST to assign genus and species-level taxonomy. Initial analysis of I. holocyclus and I. ricinus identified that > 95 % of the 16S sequences recovered belonged to the tick intracellular endosymbiont “Candidatus Midichloria mitochondrii” (CMM). A CMM-specific blocking primer was designed that decreased CMM sequences by approximately 96 % in both tick species and significantly increased the total detectable bacterial diversity, allowing identification of medically important bacterial pathogens that were previously masked by CMM.ResultsBorrelia burgdorferi sensu lato was identified in German I. ricinus, but not in Australian I. holocyclus ticks. However, bacteria of medical significance were detected in I. holocyclus ticks, including a Borrelia relapsing fever group sp., Bartonella henselae, novel “Candidatus Neoehrlichia” spp., Clostridium histolyticum, Rickettsia spp., and Leptospira inadai.ConclusionsAbundant bacterial endosymbionts, such as CMM, limit the effectiveness of next-generation 16S bacterial community profiling in arthropods by masking less abundant bacteria, including pathogens. Specific blocking primers that inhibit endosymbiont 16S amplification during PCR are an effective way of reducing this limitation. Here, this strategy provided the first evidence of a relapsing fever Borrelia sp. and of novel “Candidatus Neoehrlichia” spp. in Australia. Our results raise new questions about tick-borne pathogens in I. holocyclus ticks.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-0958-3) contains supplementary material, which is available to authorized users.

Russian Family with X-linked Alport Syndrome and Cardiovascular Abnormalities

Background: Alport syndrome (AS) is an inherited disease. Clinical findings usually include hematuria, proteinuria and arterial hypertension. Hearing loss and ocular abnormalities are common symptoms. In the last years there are some clinical observations of arterial disease (aortic aneurysm, aortic dissection) in males with AS. Methods: coding exons and splice sites of the abovementioned genes were sequenced in on Ion Torrent platform. Detailed clinical examination was obtained. Ophthalmologic and otologic evaluations, pure tone audiometry, electrocardiography, ECHO-cardiography, 24-hour blood pressure monitoring and retroperitoneal ultrasonography were performed. Results: a novel dominant missense mutation c.G3098A, p.1033 G>D in the collagen type IV alpha-5 gene (COL4A5) was revealed in five family members. New aspects of phenotype evaluated: cardiovascular abnormalities (asymptomatic aortic enlargement, left ventricular dilatation, aortic insufficiency); early childhood onset of systemic hypertension; absents of ocular abnormalities. Microhematuria, proteinuria (in progress up to 2,0 g/l) and early childhood onset of systemic hypertension are discussed as a result of heterozygous COL4A5 mutations in affected females. Conclusions: molecular genetics help to understand variable clinical phenotypes of AS patients. New findings about cardiovascular abnormalities should stimulate doctors to make cardiology examination in all affected subjects.

Abstract P5-05-07: Elucidating molecular resistance to trastuzumab using next generation sequencing in isogenic cell models

Abstract A minority of all breast cancers will express increased levels of the ERBB2 protein. They are eligible for trastuzumab-based therapy. Some will respond, but all will progress. Thus, the problem of resistance to trastuzumab has generated an urgent need to determine the underlying mechanisms of that resistance. Cancer is a genetic disease and the mechanism of trastuzumab resistance is likely also genetic in nature. However, the significant genomic heterogeneity between and within patient tumors greatly complicates the identification of a genetic mechanism of resistance for trastuzumab. In order to overcome some of these challenges, we looked to an isogenic model of trastuzumab resistance. We acquired the trastuzumab-sensitive breast cancer cell line, BT474 and two clones of this cell line that were conditioned to exhibit trastuzumab resistance. To investigate a possible genetic mechanism of trastuzumab-resistance, we performed whole exome sequencing using Ampliseq chemistry on the Ion Torrent platform from Life Technologies and paired-end RNA-sequencing on the Illumina HiSeq platform. Next-generation sequencing data was bioinformatically analyzed using tools that allowed us to filter relevant variants based on statistical and functional significance. Variants of interest were those that that arose during drug treatment, which were identified as those in each of the resistant clones, which were novel compared to the parent clone. Proteins from whole cell lysates were resolved in two dimensions using 3-10 nonlinear strips for isoelectric focusing followed by resolution via 4-20% SDS-PAGE. Proteins were visualized via Gelcode blue and cored with a biopsy punch, trypsinized, and submitted for protein ID on an LTQ XL mass spectrometer. We performed functional validation of genetic alterations through the use of somatic cell gene targeting of an ERBB2-expressing clone of the MCF-10A cell lines, a non-tumorigenic model of breast cancer, which is sensitive to trastuzumab. Exome sequencing initially yielded more than 10,000 unique DNA variants across the three clones, which after bioinformatic analysis resulted in ∼1000 variants of interest. Two-dimensional gel electrophoresis revealed 25-30 differentially expressed proteins per sample. Correlation between the sequencing and proteomics data provided us with a candidate gene list of less than 100 genes and several cellular pathways related to growth signaling and immunity. Genetic alterations were tested for their ability to cause trastuzumab resistance in MCF-10A clones. We describe herein a detailed molecular analysis for a model of trastuzumab resistance. Validated genetic alterations will be investigated in a unique collection of archival specimens, which we hope will open the path towards the development of novel agents to augment the effects of trastuzumab. Citation Format: Abde M Abukhdeir, Matthew Najor, Sanja Turturro, Melissa R Pergande, Jeffrey A Borgia, Hanif G Khalak, Melody Cobleigh. Elucidating molecular resistance to trastuzumab using next generation sequencing in isogenic cell models [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-05-07.

Abstract P5-10-07: The LiquidBiopsy in metastatic breast cancer (MBC): A novel diagnostic platform for next generation sequencing (NGS) of circulating tumor cells (CTCs)

Abstract Background: Effective treatments for advanced breast cancer are limited leaving palliative care as the only option for patients. The development of effective treatments has been challenging in part due to the biologic heterogeneity of the disease. The challenges to improve patient outcomes are represented by the availability of diagnostic technologies allowing real-time access to the genetic drivers of disease in each individual patients and the ability to match such information with novel targeted therapies. Blood based tests have a long history of providing real-time evidence in medicine. Thus blood derived templates could fill this role in genetic diagnosis. To perform, any technology for molecular analysis of tumor cells from blood must overcome limiting amounts of template and high signal to noise. Current efforts are directed to develop blood based tests for circulating biomarkers that can describe the molecular changes in breast cancer. We investigated the ability to productively sequence tumor cells recovered from whole blood. Methods: EpCAM expressing Circulating tumor cells (CTCs) were recovered from 49 patients with metastatic breast cancer on the LiquidBiopsy™ platform (Cynvenio Biosystems, Inc). CTC events were characterized using a nuclear stain (DAPI), Cytokeratin expression and absence of CD45. DNA template recovered from this population was case control sequenced with the Life Technologies AmpliSeq targeted resequencing panel on an Ion Torrent Platform using a CLIA validated sequencing pipeline and a sensitivity of 1%. Somatic single nucleotide variants (SNV) present in CTC but not the germline were identified. Critically, the false positive score for 31 normal donors thus evaluated was zero. Results: Circulating target cells were detected in all 49 patients with metastatic BC (CTC Median 28, range 2 - 4098). Upon resequencing, shared germline polymorphisms were observed in CTC and germline populations. In contrast, SNV were detected in EpCAM enriched populations but not germline in 32% of samples (allele frequency 1.0%-49.6% with 2417-22068 x coverage). Enumeration of cytokeratin expressing cells was predictive of neither the presence nor the allele frequency of detectable SNV. Interestingly mutations in the gene for PIK3ca accounted for 20% of the mutations observed including variants at K111E, E542K, E545K, and H1047L. Mutations were also observed in TP53 (8%), and MET, FGFR2, SMAD4. Twenty samples had tissue biopsy sequence data available and of those, 11 contained mutations that were evaluable in the CTC. All but one mutation were absent in the evaluable tissue biopsy Conclusions: CTC recovered by EpCAM selection directly from blood can be sequenced by NGS. The predictive value of the CK+ phenotype as a surrogate marker for mutation bearing cells is not supported by these data. Even with a relatively narrow 50 gene target platform, cancer relevant SNVs were detected in &amp;gt;30% of the patients, making targeted NGS of CTCs a promising approach for biomarker discovery and validation in MBC. Citation Format: William Strauss, Jessamine Winer-Jones, Laura Austin, Paul W Dempsey, Massimo Cristofanilli. The LiquidBiopsy in metastatic breast cancer (MBC): A novel diagnostic platform for next generation sequencing (NGS) of circulating tumor cells (CTCs) [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-10-07.

Improved transposon-based library preparation for the Ion Torrent platform.

A transposon-based approach for the construction of sequencing libraries is an efficient way of preparing samples for processing on both Illumina and Ion Torrent platforms. However, PCR-mediated incorporation of adaptors in tagged DNA fragments leaves behind self-complementary regions flanking the DNA fragment. These regions are capable of forming hairpin structures and, together with adaptors, create conditions for the potential formation of template heteroduplexes. These negatively affect the sequencing process on the Ion Torrent platform and can lead to a more than 3-fold decline in output data compared with sequencing of conventional libraries. To address this problem, we have developed MuPlus, a transposon-based protocol for barcoded library preparation for Ion Torrent, in which one adaptor is integrated by PCR and the second is integrated by ligation as a single-stranded oligonucleotide after enzymatic cleavage of a complementary part on one strand of the tag. The resulting library does not contain self-complementary, hairpin-forming regions, is free of heteroduplexes, and can be analyzed on the Ion Torrent platform with the same efficiency as a library created with a ligation-based protocol.

MP45-15 MIR-137 INHIBITED SP1 PROTEIN TRANSLATION AND MEDIATED ANTI-CANCER ACTIVITY OF ISORHAPONTIGENIN (ISO)

INTRODUCTION AND OBJECTIVES: Given the importance of surveillance in the management of non muscle invasive bladder cancer (NMIBC), noninvasive detection of circulating or urine tumor DNA (utDNA) may improve and individualize the management of bladder cancer patients. Here we evaluate methylation-specific polymerase chain reaction (MS-PCR) and two next generation sequencing (NGS) approaches to identify the presence and association of utDNA with bladder cancer recurrence. METHODS: Urine was collected from 5 patients at the time of initial diagnosis of bladder cancer (positives), from 2 patients with prior NMIBC at the time of negative surveillance cystoscopy and from 1 patient pre-repeat transurethral resection with normal biopsy (total 3 negatives). DNA was isolated from fixed paraffin embedded primary tumors (FPET) from the eight patients 4 high grade and 4 low grade e and from matched blood buffy coat cells, urine supernatant and urine sediment. Targeted NGS (covering <20,000bp) was performed on Ion Torrent (amplicon enrichment) and Illumina (hybridization capture) platforms, and MS-PCR performed with a panel of 13 assays. Percent utDNA was estimated from the ratio (urine:tumor) of single nucleotide variants (SNVs) or methylation sites compared to buffy coat. RESULTS: Tumor specific differentially methylated regions (DMRs) were found in 7 of the 8 FPETs compared to buffy coat. Tumor specific SNVs were found in 8/8 (Ion Torrent) and 6/8 (Illumina) FPETs compared to buffy coat. utDNA was found in 4 of the 5 positives by MSPCR. There was complete concordance in all positive/negative calls for utDNA between the 2 NGS platforms, with utDNA found in 4 of the 5 positives and 0 of the 3 negatives. There was complete concordance in all positive/negative calls and estimates of tumor fraction between urine supernatant and urine sediment (within patient/technology). CONCLUSIONS: Tumor specific DMRs and/or somatic SNVs were detected in 100% of NMIBC tumors using a MS-PCR gene panel and targeted NGS. Most events in utDNA were also observed in NMIBC tumor tissue at the time of diagnosis. These results provide early evidence that genetic and epigenetic events are evident in utDNA and reflect the presence and genetics of the NMIBC.

MP45-14 COMPARISON OF DIFFERENT ANALYTICAL PLATFORMS FOR THE IDENTIFICATION OF TUMOR DNA IN THE URINE OF BLADDER CANCER PATIENTS

You have accessJournal of UrologyBladder Cancer: Basic Research II1 Apr 2015MP45-14 COMPARISON OF DIFFERENT ANALYTICAL PLATFORMS FOR THE IDENTIFICATION OF TUMOR DNA IN THE URINE OF BLADDER CANCER PATIENTS Matthew J. Resnick, Richard D. Abramson, Carl Yoshizawa, Po N. Lam, Neal Shore, and Haluk Tezcan Matthew J. ResnickMatthew J. Resnick More articles by this author , Richard D. AbramsonRichard D. Abramson More articles by this author , Carl YoshizawaCarl Yoshizawa More articles by this author , Po N. LamPo N. Lam More articles by this author , Neal ShoreNeal Shore More articles by this author , and Haluk TezcanHaluk Tezcan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.1512AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Given the importance of surveillance in the management of non−muscle invasive bladder cancer (NMIBC), noninvasive detection of circulating or urine tumor DNA (utDNA) may improve and individualize the management of bladder cancer patients. Here we evaluate methylation-specific polymerase chain reaction (MS-PCR) and two next generation sequencing (NGS) approaches to identify the presence and association of utDNA with bladder cancer recurrence. METHODS Urine was collected from 5 patients at the time of initial diagnosis of bladder cancer (positives), from 2 patients with prior NMIBC at the time of negative surveillance cystoscopy and from 1 patient pre-repeat transurethral resection with normal biopsy (total 3 negatives). DNA was isolated from fixed paraffin embedded primary tumors (FPET) from the eight patients - 4 high grade and 4 low grade – and from matched blood buffy coat cells, urine supernatant and urine sediment. Targeted NGS (covering <20,000bp) was performed on Ion Torrent (amplicon enrichment) and Illumina (hybridization capture) platforms, and MS-PCR performed with a panel of 13 assays. Percent utDNA was estimated from the ratio (urine:tumor) of single nucleotide variants (SNVs) or methylation sites compared to buffy coat. RESULTS Tumor specific differentially methylated regions (DMRs) were found in 7 of the 8 FPETs compared to buffy coat. Tumor specific SNVs were found in 8/8 (Ion Torrent) and 6/8 (Illumina) FPETs compared to buffy coat. utDNA was found in 4 of the 5 positives by MS-PCR. There was complete concordance in all positive/negative calls for utDNA between the 2 NGS platforms, with utDNA found in 4 of the 5 positives and 0 of the 3 negatives. There was complete concordance in all positive/negative calls and estimates of tumor fraction between urine supernatant and urine sediment (within patient/technology). CONCLUSIONS Tumor specific DMRs and/or somatic SNVs were detected in 100% of NMIBC tumors using a MS-PCR gene panel and targeted NGS. Most events in utDNA were also observed in NMIBC tumor tissue at the time of diagnosis. These results provide early evidence that genetic and epigenetic events are evident in utDNA and reflect the presence and genetics of the NMIBC. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e541 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Matthew J. Resnick More articles by this author Richard D. Abramson More articles by this author Carl Yoshizawa More articles by this author Po N. Lam More articles by this author Neal Shore More articles by this author Haluk Tezcan More articles by this author Expand All Advertisement Advertisement PDF DownloadLoading ...

Whole genome detection of rotavirus mixed infections in human, porcine and bovine samples co-infected with various rotavirus strains collected from sub-Saharan Africa

Group A rotaviruses (RVA) are among the main global causes of severe diarrhea in children under the age of 5years. Strain diversity, mixed infections and untypeable RVA strains are frequently reported in Africa. We analysed rotavirus-positive human stool samples (n=13) obtained from hospitalised children under the age of 5years who presented with acute gastroenteritis at sentinel hospital sites in six African countries, as well as bovine and porcine stool samples (n=1 each), to gain insights into rotavirus diversity and evolution. Polyacrylamide gel electrophoresis (PAGE) analysis and genotyping with G-(VP7) and P-specific (VP4) typing primers suggested that 13 of the 15 samples contained more than 11 segments and/or mixed G/P genotypes. Full-length amplicons for each segment were generated using RVA-specific primers and sequenced using the Ion Torrent and/or Illumina MiSeq next-generation sequencing platforms. Sequencing detected at least one segment in each sample for which duplicate sequences, often having distinct genotypes, existed. This supported and extended the PAGE and RT-PCR genotyping findings that suggested these samples were collected from individuals that had mixed rotavirus infections. The study reports the first porcine (MRC-DPRU1567) and bovine (MRC-DPRU3010) mixed infections. We also report a unique genome segment 9 (VP7), whose G9 genotype belongs to lineage VI and clusters with porcine reference strains. Previously, African G9 strains have all been in lineage III. Furthermore, additional RVA segments isolated from humans have a clear evolutionary relationship with porcine, bovine and ovine rotavirus sequences, indicating relatively recent interspecies transmission and reassortment. Thus, multiple RVA strains from sub-Saharan Africa are infecting mammalian hosts with unpredictable variations in their gene segment combinations. Whole-genome sequence analyses of mixed RVA strains underscore the considerable diversity of rotavirus sequences and genome segment combinations that result from a complex evolutionary history involving multiple host species.

Comparison of Sanger and next generation sequencing performance for genotyping Cryptosporidium isolates at the 18S rRNA and actin loci

Cryptosporidium is an important enteric pathogen that infects a wide range of humans and animals. Rapid and reliable detection and characterisation methods are essential for understanding the transmission dynamics of the parasite. Sanger sequencing, and high-throughput sequencing (HTS) on an Ion Torrent platform, were compared with each other for their sensitivity and accuracy in detecting and characterising 25 Cryptosporidium-positive human and animal faecal samples.Ion Torrent reads (n = 123,857) were obtained at both 18S rRNA and actin loci for 21 of the 25 samples. Of these, one isolate at the actin locus (Cattle 05) and three at the18S rRNA locus (HTS 10, HTS 11 and HTS 12), suffered PCR drop-out (i.e. PCR failures) when using fusion-tagged PCR. Sanger sequences were obtained for both loci for 23 of the 25 samples and showed good agreement with Ion Torrent-basedgenotyping. Two samples both from pythons (SK 02 and SK 05) produced mixed 18S and actin chromatograms by Sanger sequencing but were clearly identified by Ion Torrent sequencing as C. muris. One isolate (SK 03) was typed as C. muris by Sanger sequencing but was identified as a mixed C. muris and C. tyzzeri infection by HTS. 18S rRNA Type B sequences were identified in 4/6 C. parvum isolates when deep sequenced but were undetected in Sanger sequencing.Sanger was cheaper than Ion Torrent when sequencing a small numbers of samples, but when larger numbers of samples are considered (n = 60), the costs were comparative. Fusion-tagged amplicon based approaches are a powerful way of approaching mixtures, the only draw-back being the loss of PCR efficiency on low-template samples when using primers coupled to MID tags and adaptors. Taken together these data show that HTS has excellent potential for revealing the “true” composition of species/types in a Cryptosporidium infection, but that HTS workflows need to be carefully developed to ensure sensitivity, accuracy and contamination are controlled.

Congenital hyperinsulinism in Chinese patients: 5-yr treatment outcome of 95 clinical cases with genetic analysis of 55 cases.

The aim of this study is to investigate the clinical features, therapeutic outcomes, and genetic mutations of congenital hyperinsulinism (CHI) in Chinese patients. The clinical features and therapeutic outcomes of 95 CHI cases were recorded, and genetic analyses were conducted to identify mutations in ABCC8 and KCNJ11 in 55 cases. Direct sequencing was carried out in 25 of the cases with ABCC8 and KCNJ11 mutations. Additionally, 16 samples with no mutations and the remaining 30 samples were sequenced using Ion Torrent platform. Clinical misdiagnosis occurred in 36/95 (38%) of the cases. Most (82/95; 84%) of the patients were given diazoxide therapy combined with age-dependent frequent feeding, which was effective in 54/95 (66%) cases. The side effects of diazoxide included sodium and water retention, gastrointestinal reactions, polytrichia, and thrombocytopenia. Five patients were treated with octreotide for 1-4 months, of which 80% (4/5) showed a positive response. Non-surgical therapy was effective in 71/95 (75%) cases. Of the four children who received subtotal pancreatectomy, only one had a good outcome. The remission rate of hypoglycemia was 59% for children over 2-yr-old. The CHI-related gene mutation rate was 38% for potassium channel-related genes. Early onset of CHI and a lower diazoxide response rate were associated with potassium-ATP channel gene mutations. Age-dependent frequent feeding is an acceptable therapy for CHI. Non-surgical therapy may be highly effective, in part, due to the low rate of potassium channel gene mutations. Surgical outcomes are unreliable without 18F-fluoro-L-DOPA positron emission tomography. Therefore, we do not recommend operation without definitive identification of the pathologic type.

Semiconductor-based sequencing of genome-wide DNA methylation states

Methylated DNA immunoprecipitation sequencing (MeDIP-Seq) is a widely used approach to study DNA methylation genome-wide. Here, we developed a MeDIP-Seq protocol compatible with the Ion Torrent semiconductor-based sequencing platform that is low cost, rapid, and scalable. We applied this protocol to demonstrate MeDIP-Seq on the Ion Torrent platform provides adequate coverage of CpG cytosines, the methylation states of which we validated at single-base resolution on the Infinium HumanMethylation450 BeadChip array, and accurately identifies sites of differential DNA methylation. Furthermore, we applied an integrative approach to further investigate and confirm the role of DNA methylation in alternative splicing and to profile 5mC and 5hmC variants of DNA methylation in normal human brain tissue that is localized over distinct genomic regions. These applications of MeDIP-Seq on the Ion Torrent platform have broad utility and add to the current methodologies for profiling genome-wide DNA methylation states in normal and disease conditions.

Transcriptome analysis of banana (Musa balbisiana) basedon next-generation sequencing technology

Banana (Musa spp.) is an important tropical fruit with high commercial potential. Musa balbisiana (B genome) is a progenitor of one of the most cultivated banana species and exhibits unique traits, including resistance or tolerance to many biotic and abiotic stresses. RNA sequencing of the Musa B genome would provide a vast array of transcriptomic information that could lead to the development of trait-specific markers and the discovery of new genes and regulatory sequences involved in resistance mechanisms. Thus, transcriptome sequencing was performed in Musa B genome accession Attikol using the Ion Torrent platform. This led to the generation of about 4.5 million paired-end reads, which were assembled using the MIRA assembler. The assembly produced 82,413 unique transcripts with a mean length of approximately 113 bp. The sequence similarity search against the Swiss-Prot database resulted in the identification of 35,783 unique transcripts (62.18%). Out of these, 193,826 gene ontology terms were assigned to unique transcripts. Functional annotation against PlantCYC pathway database identified 20,696 unique transcripts, which were mapped to 455 pathways. About 4780 simple sequence repeats (SSRs) were obtained from 82,413 unique transcripts. Primers could be designed for only 2628 SSRs, out of which 30 primers were randomly selected from defense-related genes to confirm their efficiency. This information will make the improvement of banana cultivars easier by facilitating the selection of resistance genes as well as the development of trait-specific markers.