Ion Torrent Personal Genome Machine Research Articles

NGS tools for traceability in candies as high processed food products: Ion Torrent PGM versus conventional PCR-cloning

The Next Generation Sequencing methodologies are considered the next step within DNA-based methods and their applicability in different fields is being evaluated. Here, we tested the usefulness of the Ion Torrent Personal Genome Machine (PGM) in food traceability analyzing candies as a model of high processed foods, and compared the results with those obtained by PCR-cloning-sequencing (PCR-CS). The majority of samples exhibited consistency between methodologies, yielding more information and species per product from the PGM platform than PCR-CS. Significantly higher AT-content in sequences of the same species was also obtained from PGM. This together with some taxonomical discrepancies between methodologies suggest that the PGM platform is still pre-mature for its use in food traceability of complex highly processed products. It could be a good option for analysis of less complex food, saving time and cost per sample.

Rapid Molecular Profiling of Myeloproliferative Neoplasms Using Targeted Exon Resequencing of 86 Genes Involved in JAK-STAT Signaling and Epigenetic Regulation

Myeloproliferative neoplasms (MPNs) are a heterogeneous group of blood disorders characterized by excess production of mature blood cells and an increased risk of late transformation to acute myeloid leukemia or primary myelofibrosis. Approximately 15% of MPN cases do not carry mutations in JAK2, CALR, or MPL and are thus often referred to as triple-negative cases. These are caused by a diverse set of rare mutations in cytokine receptors, JAK-STAT signaling pathway components, or epigenetic modifiers. In addition, some cases diagnosed as MPN are reactive rather than clonal disorders, so a negative result from a genetic screen can be informative. To obtain a comprehensive rapid molecular diagnosis for most MPNs, we developed an assay to detect genetic mutations (single nucleotide variants and/or small insertions/deletions) in 86 genes using targeted exon resequencing (AmpliSeq) and a bench-top semiconductor machine (Ion Torrent Personal Genome Machine). Our assay reliably detects well characterized mutations in JAK2, CALR, and MPL, but also rarer mutations in ASXL1, TET2, SH2B3, and other genes. Some of these mutations are novel. We find multiple mutations in advanced cases, suggesting co-operation between Janus kinase-STAT pathway mutations and epigenetic mutations in disease progression. This assay can be used to follow molecular progression, clonal heterogeneity, and drug resistance in MPNs.

Abstract 1793: POLE gene mutations confer good progression free survival in Asian women with FIGO grade 3 endometrioid endometrial carcinoma

Abstract Introduction: Endometrial carcinoma (EC) is the most common gynaecological cancer in developed countries. Mortality rates of patients with FIGO grade 3 endometrioid ECs are almost similar to that of uterine serous carcinoma and clear cell carcinoma. In 2013, the Cancer Tumour Genome Atlast (TGCA) identified POLE gene mutations in a subset of ECs, predominantly in the endometrioid subtype with high FIGO grade. While it has been suggested that POLE mutation confers good prognosis, the data remains conflicting. Our study aims to evaluate the association between the POLE gene mutations and progression free survival in Asian women with FIGO grade 3 endometrioid EC. Methods: Forty-five Asian patients diagnosed with FIGO Grade 3 endometrioid EC between 2009 and 2013 were included. Next generation sequencing (NGS) performed utilizing the Ion Torrent Personal Genome Machine (PGM) and custom designed AmpliSeq panel for formalin fixed embedded (FFPE) tissue was performed. Using the Ion Reporter, mutations with at least 10% allele frequency and present in the COSMIC database were deemed to be pathogenic. All other mutations, excluding silent mutations, were considered as variants of unknown significance (VUS). Survival curves for pathogenic somatic POLE mutated and wild-type tumors were estimated by Kaplan-Meier method and compared using logrank test. Results: All detected pathogenic and VUS POLE gene mutations were missense mutations. Our results provide further evidence to corroborate the mutation effect of POLE gene with 100% recurrence free survival in FIGO Grade 3 endometrioid EC patients with pathogenic POLE gene mutations, extends to South East Asian women as well (median follow-up POLE vs wild type; 52.5 vs 45; p = 0.2). Pathogenic somatic POLE gene mutations were present in 25.5% (12/45) patients with Grade 3 endometrioid EC. All were still alive at the time of censure with no reports of relapse following initial treatment. Three of these women harbored mutations outside exons 9 to 14 with one mutation listed as pathogenic in colorectal carcinomas. Among women without the pathogenic somatic POLE mutation, recurrence was seen in 24.2% (8/33) with 15.1% (5/33) subsequently dying of the disease. Conclusion: POLE gene mutations confer a good prognosis for FIGO grade 3 endometrioid EC and will be important in clinical decision making. POLE wild-type patients potentially show have closer follow-up and should be considered for newer targeted chemotherapeutic agents. Citation Format: Adele Wong, Chik Hong Kuick, Wai Loong Wong, Jill M. Tham, Sorsiah Mansor, Loh Eva, Sudhanshi Jain, Sock Hoai Chan, Sze Huey Tan, Shao Tzu Li, Nadkarni N. Vikas, Sung Hock Chew, Joanne Ngeow, Wanjin Hong. POLE gene mutations confer good progression free survival in Asian women with FIGO grade 3 endometrioid endometrial carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1793.

Abstract 1384: A comprehensive gene panel for precise diagnosis and treatment of childhood cancer

Abstract We have created and are validating a targeted gene panel that utilizes 20ng of FFPE DNA and 10 ng of FFPE RNA in a simple, 3-tube Ion AmpliSeq assay suitable for sequencing on the Ion Torrent platform with rapid turn around time (<3 days in lab) and automated report generation that is designed specifically to improve pediatric cancer diagnosis, identify prognostic features, and detect genomic alterations (including mutations, insertions, deletions, gene amplification, and RNA expression levels) that match current targeted therapeutic targets. The core content is identical to the Oncomine Comprehensive Assay as employed by the NCI MATCH program. In addition, a total of 128 genes known to be genomically altered in pediatric cancer are interrogated (either hotspots or coding exons), 21 genes frequently amplified in pediatric cancer, and 157 gene fusions (including over 1,500 variants) unique to these tumors are included. Unique to this panel are 22 pharmacogenomic gene polymorphisms that influence cytotoxic drug metabolism, to guide patient-specific dosing. Content was chosen to cover all common pediatric malignancies, including pediatric leukemia (ALL, AML, and multiple variants within each), lymphoma, brain tumors, neuroblastoma, and sarcomas, by a working group composed of pediatric oncologists and pathologists. Specific genomic alterations were identified from a comprehensive search of the pediatric cancer literature by the panel and categorized by level of evidence (FDA approved drug, open clinical trial, or peer-reviewed literature identifying a feature in a pediatric cancer). This panel includes 45 of the 51 gene targets identified by the COG TAP committee for pediatric cancer specific gene target-therapeutic agent matching. In contrast to the NCI MATCH program, which currently employs the Ion Torrent Personal Genome Machine and Ion 318 Chip, this panel is intended for use on the newly available Ion S5 platform, due to its significantly enlarged content. Verification of individual feature identification by orthogonal assays (Sanger sequencing, PCR, FISH, SNP arrays) is in process. The assay will be released initially as an LDP, with intended updated content as new features and therapeutics are identified. Unlike current panels for specific features like single gene mutations or known gene translocations, this panel is intended to detect all currently known genomic alterations with level I, II, or III evidence in pediatric cancer, as well as the ability to detect new gene mutations and fusion variants (subject to secondary assay validation). Also unique is the ability to measure gene amplification and RNA expression levels in one comprehensive panel starting with 30 ng or less of nucleic acid extracted from routine clinical FFPE specimens. This is the only comprehensive gene panel for pediatric cancer that encompasses diagnostic, prognostic, pharmacogenomic, and therapeutic targets in one assay. Citation Format: Timothy J. Triche, Jackie Biegel, Alex Judkins, Tracy Busse, Alan Wayne, Deepa Bhojwani, Shahab Asgharzadeh, Matthew Oberley, David Parham. A comprehensive gene panel for precise diagnosis and treatment of childhood cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1384.

Targeted Sequencing of Myogenic Regulatory Factors and Myostatins Reveals an Association between MSTN‐1 and Interorbital Distance in Orange‐spotted Grouper, Epinephelus coioides (Hamilton, 1822)

AbstractThe aim of this study was to investigate single nucleotide polymorphisms (SNPs) in genes that are presumed to control muscle growth and to determine their potential association with growth in a cultured population of orange‐spotted grouper, Epinephelus coioides. Seven genes, myogenic regulatory factor 4 (MRF4), Myf5, MSTN‐1, MSTN‐2, MyoD1, MyoD2, and myogenin, were selected for the investigation, covering approximately 26 kbp. First, the two clades for the genes MSTN (myostatin) and MyoD were confirmed in this species using Bayesian inference analysis of the phylogenetic relationships. Then, the seven genes were enriched by polymerase chain reaction and sequenced using an Ion Torrent Personal Genome Machine. A total of 586 SNPs were discovered. Linkage disequilibrium was decayed by 50% within 250 bp based on the combined data, which means that there was high resolution in the association mapping. A mixed linear model considering the population structure and kinship was used to detect the associations between genotypes and phenotypes. Only one site (KR269814.1:g.22T>G) in MSTN‐1 was found to be significantly associated with a measured trait, the interorbital distance (false discovery ratio < 0.05), and it explained 12.4% of the phenotype variation of this trait. This study provides insight on strategies for molecular marker‐assisted breeding in orange‐spotted grouper.

Hot spot mutations in Finnish non-small cell lung cancers

ObjectivesNon-small cell lung cancer (NSCLC) is a common cancer with a poor prognosis. The aim of this study was to screen Finnish NSCLC tumor samples for common cancer-related mutations by targeted next generation sequencing and to determine their concurrences and associations with clinical features. Materials and methodsSequencing libraries were prepared from DNA isolated from formalin-fixed, paraffin-embedded tumor material of 425 patients using the AmpliSeq Colon and Lung panel covering mutational hot spot regions of 22 cancer genes. Sequencing was performed with the Ion Torrent Personal Genome Machine (PGM). ResultsData analysis of the hot spot mutations revealed mutations in 77% of the patients, with 7% having 3 or more mutations reported in the Catalogue of Somatic Mutations in Cancer (COSMIC) database. Two of the most frequently mutated genes were TP53 (46%) and KRAS (25%). KRAS codon 12 mutations were the most recurrently occurring mutations. EGFR mutations were significantly associated with adenocarcinoma, female gender and never/light-smoking history; CTNNB1 mutations with light ex-smokers, PIK3CA and TP53 mutations with squamous cell carcinoma, and KRAS with adenocarcinoma. TP53 mutations were most prevalent in current smokers and ERBB2, ERBB4, PIK3CA, NRAS, NOTCH1, FBWX7, PTEN and STK11 mutations occurred exclusively in a group of ever-smokers, however the association was not statistically significant. No mutation was found that associated with asbestos exposure. ConclusionFinnish NSCLC patients have a similar mutation profile as other Western patients, however with a higher frequency of BRAF mutations but a lower frequency of STK11 and ERBB2 mutations. Moreover, TP53 mutations occurred frequently with other gene mutations, most commonly with KRAS, MET, EGFR and PIK3CA mutations.

First application of next-generation sequencing in Moroccan breast/ovarian cancer families and report of a novel frameshift mutation of the BRCA1 gene

At present, breast cancer is the most common type of cancer in females. The majority of cases are sporadic, but 5-10% are due to an inherited predisposition to develop breast and ovarian cancers, which are transmitted as an autosomal dominant form with incomplete penetrance. The beneficial effects of clinical genetic testing, including next generation sequencing (NGS) for BRCA1/2 mutations, is major; in particular, it benefits the care of patients and the counseling of relatives that are at risk of breast cancer, in order to reduce breast cancer mortality. BRCA genetic testing was performed in 15 patients with breast cancer and a family with positivity for the heterozygous c.6428C>A mutation of the BRCA2 gene. Informed consent was obtained from all the subjects. Genomic DNAs were extracted and the NGS for genes was performed using the Ion Torrent Personal Genome Machine (PGM) with a 316 chip. The reads were aligned with the human reference HG19 genome to elucidate variants in the BRCA1 and BRCA2 genes. Mutations detected by the PGM platform were confirmed by target direct Sanger sequencing on a second patient DNA sample. In total, 4 BRCA variants were identified in 6 families by NGS. Of these, 3 mutations had been previously reported: c.2126insA of BRCA1, and c.1310_1313delAAGA and c.7235insG of BRCA2. The fourth variant, c.3453delT in BRCA1, has, to the best of our knowledge, never been previously reported. The present study is the first to apply NGS of the BRCA1 and BRCA2 genes to a Moroccan population, prompting additional investigation into local founder mutations and variant characteristics in the region. The variants with no clear clinical significance may present a diagnostic challenge when performing targeted resequencing. These results confirm that an NGS approach based on Ampliseq libraries and PGM sequencing is a highly efficient, speedy and high-throughput mutation detection method, which may be preferable in lower income countries.

Haplotype diversity in mitochondrial genome in a Chinese Han population.

Investigations into the use of mitochondrial genome (mtGenome) typing by massively parallel sequencing technologies are well underway in many areas, including forensic genetics. Previous studies have demonstrated that mtGenome sequencing data generated from Ion torrent personal genome machine (PGM) system were highly viable and reliable in forensic research. In this study, 145 whole mtGenomes from unrelated Chinese Han population were sequenced using the Ion PGM system. Results showed that 145 distinct haplotypes were obtained at a relatively high coverage with limited strand bias. The distribution of variants across the entire mtGenomes was illustrated and 70.74% of the variants were observed outside of the control region. An overall increase in the number of unique haplotypes as well as haplotype diversity were observed by detection of mtGenome compared with hypervariable region I/II (HV I/II) and control region (CR). This study demonstrates the substantially higher degree of haplotype resolution with whole-mtGenome sequences in comparison to HV I/II or CR that historically targeted for forensic testing, which shows the potential value of mtGenome typing in forensic testing in the future.

Advances and Applications of Ion Torrent Personal Genome Machine in Cutaneous Squamous Cell Carcinoma Reveal Novel Gene Mutations.

The Ion Torrent Personal Genome Machine (Ion PGM) is a semiconductor-based sequencing technology that is high quality, scalable, and economic. Its applications include genomic sequencing, drug resistance testing, microbial characterization, and targeted sequencing in cancer studies. However, little is known about the application of Ion PGM in cutaneous squamous cell carcinoma (cSCC). We therefore investigated the utility and validity of Ion PGM in cSCC and also gained a better understanding of the underlying molecular biology of cSCC. We detected novel gene mutations (KDR, FGFR2, and EGFR) in two cSCC patients. Moreover, we validated these mutations by pyrosequencing and Sanger sequencing. Our results indicated that the mutation screen using Ion PGM is consistent with traditional sequencing methods. Notably, these identified mutations were present at significantly higher rates in high-risk cSCC. Our results demonstrate a method to detect targetable genes in high-risk cSCC, and suggest that Ion PGM may enable therapeutic decision-making and future potential targets for personalized therapies in cSCC.

Genetic Mutation Analysis of Parkinson's Disease Patients Using Multigene Next-Generation Sequencing Panels.

Parkinson's disease (PD) is the second most common neurodegenerative disorder, affecting millions of people. Genome-wide association studies (GWAS) have found>25 genetic risk factors and at least 15 loci directly associated with PD. Recent advances in new next-generation DNA sequencing technologies, such as the semiconductor-based Ion Torrent platform, make multigene sequencing cheaper, faster, and more reliable. Our objective was to test the power of this next-generation sequencing technology to analyze large samples by screening the majority of the most relevant PD-related genes known for single and compound mutations. To archive a rapid, robust, and cost-effective genetic analysis of a PD cohort, we designed a multiplex, polymerase chain reaction (PCR)-based primer panel to amplify and sequence coding exons of 15 PD-associated genes (SNCA, LRRK2, PARK2, PINK1, PARK7, GIGYF2, ATP13A2, UCHL1, PLA2G6, FBXO7, EIF4G1, VPS35, ACMSD, APOE, and GBA). We conducted parallel sequencing using the Ion Torrent Personal Genome Machine(®) system to detect mutations in 92 blood DNA samples from PD patients. After bioinformatics analysis and filtering, 95.13% coverage of the targeted region was obtained at>40-fold mean coverage. The results revealed 44 previously documented variants in these 15 genes, with five revealed as pathogenic. We also discovered six novel variants, five of which had an in silico prediction of being pathogenic. Benchtop next-generation sequencing is a powerful method for genetic screening for PD. Our results indicated that it yielded a high frequency of discovery (66%; n=92) of variants in carriers from an enriched Spanish PD sample.

Identification of novel mutations including a double mutation in patients with inherited cardiomyopathy by a targeted sequencing approach using the Ion Torrent PGM system.

Inherited cardiomyopathy is the major cause of sudden cardiac death (SCD) and heart failure (HF). The disease is associated with extensive genetic heterogeneity; pathogenic mutations in cardiac sarcomere protein genes, cytoskeletal protein genes and nuclear envelope protein genes have been linked to its etiology. Early diagnosis is conducive to clinical monitoring and allows for presymptomatic interventions as needed. In the present study, the entire coding sequences and flanking regions of 12 major disease (cardiomyopathy)-related genes [namely myosin, heavy chain 7, cardiac muscle, β (MYH7); myosin binding protein C, cardiac (MYBPC3); lamin A/C (LMNA); troponin I type 3 (cardiac) (TNNI3); troponin T type 2 (cardiac) (TNNT2); actin, α, cardiac muscle 1 (ACTC1); tropomyosin 1 (α) (TPM1); sodium channel, voltage gated, type V alpha subunit (SCN5A); myosin, light chain 2, regulatory, cardiac, slow (MYL2); myosin, heavy chain 6, cardiac muscle, α (MYH6); myosin, light chain 3, alkali, ventricular, skeletal, slow (MYL3); and protein kinase, AMP-activated, gamma 2 non-catalytic subunit (PRKAG2)] in 8 patients with dilated cardiomyopathy (DCM) and in 8 patients with hypertrophic cardiomyopathy (HCM) were amplified and then sequenced using the Ion Torrent Personal Genome Machine (PGM) system. As a result, a novel heterozygous mutation (MYH7, p.Asn885Thr) and a variant of uncertain significance (TNNT2, p.Arg296His) were identified in 2 patients with HCM. These 2 missense mutations, which were absent in the samples obtained from the 200 healthy control subjects, altered the amino acid that was evolutionarily conserved among a number of vertebrate species; this illustrates that these 2 non-synonymous mutations play a role in the pathogenesis of HCM. Moreover, a double heterozygous mutation (PRKAG2, p.Gly100Ser plus MYH7, p.Arg719Trp) was identified in a patient with severe familial HCM, for the first time to the best of our knowledge. This patient provided us with more information regarding the genotype-phenotype correlation between mutations of MYH7 and PRKAG2. Taken together, these findings provide insight into the molecular mechanisms underlying inherited cardiomyopathy. The mutations identified in this study may be further investigated in the future in order to improve the diagnosis and treatment of patients with inherited cardiomyopathy. Furthermore, our findings indicated that sequencing using the Ion Torrent PGM system is a useful approach for the identification of pathogenic mutations associated with inherited cardiomyopathy, and it may be used for the risk evaluation of individuals with a possible susceptibility to inherited cardiomyopathy.

Evaluation of the effects of intrapartum antibiotic prophylaxis on newborn intestinal microbiota using a sequencing approach targeted to multi hypervariable 16S rDNA regions.

Different factors are known to influence the early gut colonization in newborns, among them the perinatal use of antibiotics. On the other hand, the effect on the baby of the administration of antibiotics to the mother during labor, referred to as intrapartum antibiotic prophylaxis (IAP), has received less attention, although routinely used in group B Streptococcus positive women to prevent the infection in newborns. In this work, the fecal microbiota of neonates born to mothers receiving IAP and of control subjects were compared taking advantage for the first time of high-throughput DNA sequencing technology. Seven different 16S rDNA hypervariable regions (V2, V3, V4, V6+V7, V8, and V9) were amplified and sequenced using the Ion Torrent Personal Genome Machine. The results obtained showed significant differences in the microbial composition of newborns born to mothers who had received IAP, with a lower abundance of Actinobacteria and Bacteroidetes as well as an overrepresentation of Proteobacteria. Considering that the seven hypervariable regions showed different discriminant ability in the taxonomic identification, further analyses were performed on the V4 region evidencing in IAP infants a reduced microbial richness and biodiversity, as well as a lower number of bacterial families with a predominance of Enterobacteriaceae members. In addition, this analysis pointed out a significant reduction in Bifidobacterium spp. strains. The reduced abundance of these beneficial microorganisms, together with the increased amount of potentially pathogenic bacteria, may suggest that IAP infants are more exposed to gastrointestinal or generally health disorders later in age.

Abstract P6-07-03: Genetic heterogeneity revealed by targeted exome sequencing in advanced triple-negative breast cancer

Abstract Introduction: Clinical decisions in oncology are increasingly guided by molecular genetics. Next-generation sequencing (NGS) provides a platform for identifying potential therapeutic targets and clinically relevant genetic markers for personalized therapy. Methodology: We performed sequencing with the SmartGen 421 NGS gene panel, on relapsed triple negative breast cancer (TNBC) specimens (n = 11) by obtaining a biopsy from the most recent site of progressive disease. Custom HaloplexTM reagents were used to capture the regions of interest by hybridization to probes corresponding to target regions. Targets were amplified to enrich DNA libraries. Libraries were sequenced on an Ion Torrent Personal Genome Machine, and sequences compared to the reference genome GRCh37/hg19. The coding regions and +/-5 base pairs of the introns of 421 genes were sequenced. Analytic sensitivity for SNP calls was 94.1% (69.2%-99.7%, at 95% CI), specificity was 100%, for introns and deletions sensitivity was 78.9% (62.2%-89.9%, 95% CI) and specificity was 99.994%. Results: A total of 96 genes contained genetic variants, with each case harbouring 4 to 15 variants. No two cases had identical mutation profiles, indicating significant genetic heterogeneity. We tabulated a) all the genetic variants and b) potentially actionable variants based on predicted effects on protein structure and conservation of mutated amino acids, using COSMIC. Among the identified variants, n = 10/96 have been previously described in COSMIC. The others are previously unreported, de novo mutations of unknown functional significance. Not surprisingly, n = 8/11 tumors contained TP53 mutations, 7 of which previously described. Genes involved in DNA repair and cell stress responses were found frequently mutated (e.g., ATM, ATR, CHEK1, CHEK2, MLH1, MUTYH, PMS1, PMS2, XPC). Interestingly, 5 mutations in the PI3K-AKT-TOR pathway were identified, including two previously described PIK3CA mutations historically linked to estrogen receptor positive disease. We did not observe PTEN mutations often linked to basal-like breast cancer. Among identified genetic variants, n = 23/96 affected genes encoding kinases (receptor and non-receptor tyrosine kinases, serine/threonine kinases, lipid kinases), including 4 involved in DNA damage response. Developmental genes in the Notch, Wnt and Hedgehog pathways were also mutated. Our results emphasize molecular heterogeneity of TNBC and suggest a variety of possible mechanisms for treatment resistance. We must also acknowledge limitation of this study, which include: lack of information on transcript expression levels of the mutated genes, the limited set of genes examined (n = 421/ case), incomplete knowledge of functional and structural effects of genetic variants on each target gene and the contextual effect of coexisting variants, and the possibility that rare cellular clones may be below the limit of detection. Conclusions: We demonstrate that targeted sequencing using NGS is cost-effective, reliable and feasible for clinical use. Multiparamenter molecular profiling (DNA, RNA, protein, metabolism) will eventually provide more complete pictures of individual tumors for truly personalized treatment. Citation Format: Pannuti A, Filipovic A, Hicks C, Lefkowitz E, Ptacek T, Miele L, Stebbing J. Genetic heterogeneity revealed by targeted exome sequencing in advanced triple-negative breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-07-03.

Recurrent mutation in the crystallin alpha A gene associated with inherited paediatric cataract

BackgroundCataract is a major cause of childhood blindness worldwide. The purpose of this study was to determine the genetic cause of paediatric cataract in a South Australian family with a bilateral lamellar paediatric cataract displaying variable phenotypes.Case presentationFifty-one genes implicated in congenital cataract in human or mouse were sequenced in an affected individual from an Australian (Caucasian) family using a custom Ampliseq library on the Ion Torrent Personal Genome Machine. Reads were mapped against the human genome (hg19) and variants called with the Torrent Suite software. Variants were annotated to dbSNP 137 using Ion Reporter (IR 1.6.2) and were prioritised for validation if they were novel or rare and were predicted to be protein changing. We identified a previously reported oligomerization disrupting mutation, c.62G > A (p.R21Q), in the Crystallin alpha A (CRYAA) gene segregating in this three generation family. No other novel or rare coding mutations were detected in the known cataract genes sequenced. Microsatellite markers were used to compare the haplotypes between the family reported here and a previously published family with the same segregating mutation. Haplotype analysis indicated a potential common ancestry between the two South Australian families with this mutation. The work strengthens the genotype-phenotype correlations between this functional mutation in the crystallin alpha A (CRYAA) gene and paediatric cataract.ConclusionThe p.R21Q mutation is the most likely cause of paediatric cataract in this family. The recurrence of this mutation in paediatric cataract families is likely due to a familial relationship.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-016-1890-0) contains supplementary material, which is available to authorized users.

The next generation of metastatic melanoma: uncovering the genetic variants for anti-BRAF therapy response.

Metastatic melanoma (MM) is a highly aggressive cancer with a median overall survival of 6–9 months, notwithstanding the numerous efforts in development of new therapeutic approaches. To this aim we tested the clinical applicability of the Ion Torrent Personal Genome Machine to simultaneously screen MM patients in order to individuate new or already known SNPs and mutations able to predict the duration of response to BRAF inhibitors. An Ampliseq Custom Panel, including 11 crucial full length genes involved in melanoma carcinogenesis and therapy response pathways, was created and used to analyze 25 MM patients. We reported BRAFV600 and NRASQ61 mutations in 68% and 24% of samples, respectively. Moreover, we more frequently identified the following alterations related to BRAF status: PIK3CAI391M (44%) and KITD737N (36%) mutations, CTLA4T17A (52%), MC1RV60L (32%) and MITFS473A (60%) polymorphisms. Considering the progression free survival (PFS), statistical analyses showed that BRAFV600 patients without any of these more frequent alterations had a higher median PFS. Protein structure changes seem to be due to these variants by in silico analysis. In conclusion, a Next-Generation Sequencing approach with custom panel may provide new information to evaluate tumor-specific therapeutic susceptibility and individual prognosis to improve the care of MM patients.

Development of an Analysis Pipeline Characterizing Multiple Hypervariable Regions of 16S rRNA Using Mock Samples.

ObjectivesThere is much speculation on which hypervariable region provides the highest bacterial specificity in 16S rRNA sequencing. The optimum solution to prevent bias and to obtain a comprehensive view of complex bacterial communities would be to sequence the entire 16S rRNA gene; however, this is not possible with second generation standard library design and short-read next-generation sequencing technology.MethodsThis paper examines a new process using seven hypervariable or V regions of the 16S rRNA (six amplicons: V2, V3, V4, V6-7, V8, and V9) processed simultaneously on the Ion Torrent Personal Genome Machine (Life Technologies, Grand Island, NY). Four mock samples were amplified using the 16S Ion Metagenomics Kit™ (Life Technologies) and their sequencing data is subjected to a novel analytical pipeline.ResultsResults are presented at family and genus level. The Kullback-Leibler divergence (DKL), a measure of the departure of the computed from the nominal bacterial distribution in the mock samples, was used to infer which region performed best at the family and genus levels. Three different hypervariable regions, V2, V4, and V6-7, produced the lowest divergence compared to the known mock sample. The V9 region gave the highest (worst) average DKL while the V4 gave the lowest (best) average DKL. In addition to having a high DKL, the V9 region in both the forward and reverse directions performed the worst finding only 17% and 53% of the known family level and 12% and 47% of the genus level bacteria, while results from the forward and reverse V4 region identified all 17 family level bacteria.ConclusionsThe results of our analysis have shown that our sequencing methods using 6 hypervariable regions of the 16S rRNA and subsequent analysis is valid. This method also allowed for the assessment of how well each of the variable regions might perform simultaneously. Our findings will provide the basis for future work intended to assess microbial abundance at different time points throughout a clinical protocol.

Early onset esophageal adenocarcinoma: a distinct molecular entity?

Esophageal adenocarcinoma (EAC) is typically diagnosed in elderly with a median age of 68 years. The incidence of EAC has been rising over the last decades, also among young adults. The aim of the study was to investigate whether early onset EAC is a distinct molecular entity. To identify early onset EACs, the nationwide network and registry of histo- and cytopathology in the Netherlands (PALGA) was searched. Twenty-eight tumors of patients aged ≤40 years were selected and matched with 27 tumors of patients aged ≥68 years. DNA was isolated from surgically resected specimen and sequenced on the Ion Torrent Personal Genome Machine with the Ion AmpliSeq Cancer Panel. No differences in mutational load between early onset and conventional EACs were observed (P=0.196). The most frequently mutated genes were TP53 (73%) and P16 (16%). Additional mutations in early onset EACs occurred exclusively in: APC, CDH1, CTNNB1, FGFR2, and STK11. In the conventional EACs additional mutations were exclusively identified in: ABL1, FBXW7, GNA11, GNAS, KRAS, MET, SMAD4, and VHL. Additional mutations besides TP53 and P16 seem to occur in different genes related to cell fate pathways for early onset EACs, while the additional mutations in conventional EACs are related to survival pathways.

Complete Genome Sequence of Corynebacterium pseudotuberculosis Viscerotropic Strain N1.

We present the complete genome sequence of Corynebacterium pseudotuberculosis strain N1. The sequencing was performed with the Ion Torrent Personal Genome Machine system. The genome is a circular chromosome with 2,337,845 bp, a G+C content of 52.85%, and a total of 2,045 coding sequences, 12 rRNAs, 49 tRNAs, and 58 pseudogenes.

Ion Torrent next-generation sequencing reveals the complete mitochondrial genome of koi carp (Cyprinus carpio, Cyprinidae)

The complete mitochondrial genome of the koi carp (Cyprinus carpio, Cyprinidae) was sequenced in the present study by using Ion Torrent Personal Genome Machine (PGM) platform for the first time. The mitochondrial genome sequence is 16 581 bp in size and consists of 13 protein-coding genes, 22 tRNA genes, two rRNA genes and one control region. The gene order and organization were similar to most of the other teleost. The nucleotide compositions of the light strand are 24.82% of A, 31.92% of T, 27.53% of G and 15.73% of C. With the exception of eight tRNA genes and the NADH dehydrogenase subunit 6 (ND6), all other mitochondrial genes are encoded on the heavy strand. The phylogenetic tree constructed using a maximum-likelihood model showed sister relationship of koi carp to other Cyprinidae fishes.

A Multiplex PCR-Based Next-Generation Sequencing Approach Has Detected a Common Large Deletion in STS Gene in a Patient with X-Linked Ichthyosis

Several nuclear genes have been found to be linked to ichthyosis, and Next Generation Sequencing approach on panels of targeted genes has turned out to be particularly useful in analyzing diseases characterized by significant genetic and phenotypic heterogeneity. We developed a panel of 26 genes to be screened with the Ion Personal Genome Machine (PGM) for causative mutations relating to ichthyosis. Sequencing runs were obtained from a patient with ichthyosis using the Ion Torrent PGM and then processed with Ion Torrent Suite, Variant Caller, Coverage Analysis and wANNOVER tools. No causative mutations were found using Variant Caller and wANNOVER softwares, whereas the “Coverage Analysis” tool revealed a common large deletion in STS gene in a patient with X-linked ichthyosis. Identification of indels in Next Generation Sequencing (NGS) data is a veritable challenge. This study demonstrates the efficacy and effectiveness of using NGS approach to detect large deletions without resorting to specific algorithms for “indel” detection. Our results indicate that the NGS panel is a useful, rapid and cost-effective screening test for patients whose features are suggestive of a genetic etiology involving one of the genes embedded in the panel. It is an excellent alternative to Sanger sequencing as for costs, ease of analysis, and turnaround time.