Ion Torrent PGM Platform Research Articles

Effect of Parkinson\u2019s disease and related medications on the composition of the fecal bacterial microbiota

Parkinson’s disease (PD) is one of the most common neurodegenerative disorders. PD patients suffer from gastrointestinal dysfunctions and alterations of the autonomous nervous system, especially its part in the gut wall, i.e., the enteric nervous system (ENS). Such alterations and functional gastrointestinal deficits often occur years before the classical clinical symptoms of PD appear. Until now, only little is known about PD-associated changes in gut microbiota composition and their potential implication in PD development. In order to increase knowledge in this field, fecal samples of 34 PD patients and 25 healthy, age-matched control persons were investigated. Here, the V4 and V5 hypervariable region of bacterial 16S rRNA genes was PCR-amplified and sequenced using an Ion Torrent PGM platform. Within the PD group, we observed a relative decrease in bacterial taxa which are linked to health-promoting, anti-inflammatory, neuroprotective or other beneficial effects on the epithelial barrier, such as Faecalibacterium and Fusicatenibacter. Both taxa were lowered in PD patients with elevated levels of the fecal inflammation marker calprotectin. In addition, we observed an increase in shares of the Clostridiales family XI and their affiliated members in these samples. Finally, we found that the relative abundances of the bacterial genera Peptoniphilus, Finegoldia, Faecalibacterium Fusicatenibacter, Anaerococcus, Bifidobacterium, Enterococcus, and Ruminococcus were significantly influenced by medication with L-dopa and entacapone, respectively. Our data confirm previously reported effects of COMT inhibitors on the fecal microbiota of PD patients and suggest a possible effect of L-dopa medication on the relative abundance of several bacterial genera.

Identification of novel BRCA1 large genomic rearrangements by a computational algorithm of amplicon-based Next-Generation Sequencing data.

BackgroundGenetic testing for BRCA1/2 germline mutations in hereditary breast/ovarian cancer patients requires screening for single nucleotide variants, small insertions/deletions and large genomic rearrangements (LGRs). These studies have long been run by Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). The recent introduction of next-generation sequencing (NGS) platforms dramatically improved the speed and the efficiency of DNA testing for nucleotide variants, while the possibility to correctly detect LGRs by this mean is still debated. The purpose of this study was to establish whether and to which extent the development of an analytical algorithm could help us translating NGS sequencing via an Ion Torrent PGM platform into a tool suitable to identify LGRs in hereditary breast-ovarian cancer patients.MethodsWe first used NGS data of a group of three patients (training set), previously screened in our laboratory by conventional methods, to develop an algorithm for the calculation of the dosage quotient (DQ) to be compared with the Ion Reporter (IR) analysis. Then, we tested the optimized pipeline with a consecutive cohort of 85 uncharacterized probands (validation set) also subjected to MLPA analysis. Characterization of the breakpoints of three novel BRCA1 LGRs was obtained via long-range PCR and direct sequencing of the DNA products.ResultsIn our cohort, the newly defined DQ-based algorithm detected 3/3 BRCA1 LGRs, demonstrating 100% sensitivity and 100% negative predictive value (NPV) (95% CI [87.6–99.9]) compared to 2/3 cases detected by IR (66.7% sensitivity and 98.2% NPV (95% CI [85.6–99.9])). Interestingly, DQ and IR shared 12 positive results, but exons deletion calls matched only in five cases, two of which confirmed by MLPA. The breakpoints of the 3 novel BRCA1 deletions, involving exons 16–17, 21–22 and 20, have been characterized.ConclusionsOur study defined a DQ-based algorithm to identify BRCA1 LGRs using NGS data. Whether confirmed on larger data sets, this tool could guide the selection of samples to be subjected to MLPA analysis, leading to significant savings in time and money.

Newly Diagnosed AML Patient Samples Demonstrate High Degree of Concordance in Identification of Pathogenic Mutations By Next Generation Sequencing (NGS) Performed at Enrolling Institutions Compared to Central Laboratory Results in the Beat AML Master Trial

Newly Diagnosed AML Patient Samples Demonstrate High Degree of Concordance in Identification of Pathogenic Mutations By Next Generation Sequencing (NGS) Performed at Enrolling Institutions Compared to Central Laboratory Results in the Beat AML Master Trial

Draft genome sequence of Escherichia coli M51-3: a multidrug-resistant strain assigned as ST131-H30 recovered from infant diarrheal infection in Mexico.

In this study, we report the draft genome sequence of a multidrug-resistant (MDR)Escherichia coli strain recovered from stool sample of an outpatient infant girl with acute diarrheal infection in Mexico. Antimicrobial susceptibility testing and PCR-based detection of diarrheagenic E. coli (DEC) were performed. In addition, genomic DNA from E. coli strain M51-3 was sequenced using Ion Torrent PGM platform with 200-bp chemistry and generated reads were de novo assembled using SPAdes v3.11. The draft genome was annotated and analyzed regarding multilocus sequence typing (MLST), serotyping, fimH typing, plasmid replicons, acquired antimicrobial resistance and virulence genes using web tools available at the Center for Genomic Epidemiology. A draft genome comprising 5 088 545 bp in length and 5308 protein-coding sequences was generated. In silico typification revealed that E. coli strain M51-3 belongs to ST131-O25:H4-H30 pandemic subclone. Several genes associated with resistance to β-lactams [blaTEM-1B], aminoglycosides [aph(3'')-Ib, aadA5, aph(6)-Id and aac(3)-IId], sulfonamides [sul1 and sul2], trimethoprim [dfrA17], and tetracycline [tet(A)] were identified. Besides, point mutations in gyrA, parC, and parE genes were detected. Interestingly, the enterotoxin-coding virulence gene senB was evidenced. To our knowledge, this is the first draft genome of an E. coli ST131-O25:H4-H30 strain recovered from infant diarrheal stool sample in Mexico. The genome sequence of E. coli M51-3 presented here will be helpful to understand the genomic diversity of this highly virulent and MDR successfully pandemic bacterial pathogen.

Analysis of 124 SNP loci included in HID Ampliseq identity panel in a small population of Rio de Janeiro, Brazil

Implementation of massively parallel sequencing platforms can bring a great contribution to Forensic Genetics field, with a great saving of time and costs, as well as allowing reliable results to be obtained from small or extremely degraded samples. The aim of this work was to analyze 124 SNP loci (90 autosomal and 34 Y-SNP) included in HID-Ion Ampliseq Identity Panel in a small sample from Rio de Janeiro state, Brazil. Samples from 12 non-related individuals were amplified with HID-Ion Ampliseq Identity Panel and sequenced on the Ion Torrent PGM platform (Thermo Fisher Scientific); genotypes were generated with HID SNP Genotyper plugin and forensic parameters were calculated with PowerStats v.12. All samples were successfully genotyped and were used to calculate allele frequencies, homozygosity, heterozygosity, random match probability (RMP) and exclusion power for all 90 autosomal SNP loci. Using the formula proposed by Budowle et al. (1996), only 4 of the 90 loci genotyped (4,4%) showed allele frequencies below the minimum required. It means that although a small set of individuals was used on this study, it may have shown a good perspective of Rio de Janeiro state allele frequencies. Among the 11 male samples analyzed, a prevalence of the haplogroup R1b of Y chromosome was observed, followed by the haplogroups E, Q and J. Such distribution reflects the results demonstrated in other studies for the population of Rio de Janeiro. All results together demonstrate the usefulness and applicability of SNP analysis on Ion Torrent PGM.

Massive parallel sequencing and osteogenesis imperfecta: An essential tool for forensic investigation over child abuse

Osteogenesis imperfecta (OI) is a rare disease of collagen synthesis causing bone fragility. Also called “glass bone disease” since it manifests as spontaneous fractures, it is classified into nine types, both with dominant and recessive transmission. In 95% of cases OI is caused by mutations in COL1A1 and COL1A2 genes encoding the alpha1 and alpha2 chains of type 1 collagen, mainly null variants caused by frame-shift/nonsense mutations or splicing defects. In infants the differential diagnosis include not-accidental trauma, so child abuse. Families suspected of abuse often provide an unverified history of frequent fractures; conversely, the family history of individuals with OI often does not reveal any other affected individuals because of a de novo pathogenic variant in the proband or the presence of a mild phenotype in relatives. Therefore, legal medicine unit with DNA lab is crucial in these cases since it could early collect living or autopsy samples when a child abuse is suspected and then test DNA. We set up a MPS (massively parallel sequencing) panel including the coding regions of COL1A1 and COL1A2 and other 11 genes known to cause OI. We presented a case of suspected abuses in 2-month-old baby. MPS libraries were sequenced by Ion Torrent PGM platform; pathogenic variants and VUS (variants of uncertain significance) were confirmed by Sanger sequencing and familial segregation study was performed to better characterize the clinical significance of the mutation. This study remarks that MPS could help not only for identification, ancestry/phenotyping or molecular autopsy applications but also for forensic investigation over child abuse. The usefulness of this assay for diagnostic projects on victims of abuse together with post-mortem cases is discussed.

Full mtDNA genome sequencing of Brazilian admixed populations: A forensic-focused evaluation of a MPS application as an alternative to Sanger sequencing methods

The use of Massive Parallel Sequencing (MPS) techniques have been proposed by the forensic community as an alternative to Sanger sequencing methods in routine forensic casework analysis regarding mitochondrial DNA (mtDNA). Interesting features of MPS include high throughput, ability to simultaneously genotype a significant number of samples by barcoding techniques, processing automation, reduced time and costs, among others. Advantages include the capability of generating full mtDNA genome sequences versus usual techniques, usually limited to hypervariable or control regions exclusively. In this work, 96 reference single-source samples from three different Brazilian cities were subjected to full mtDNA genome sequencing by MPS techniques using an early-access version of Precision ID mtDNA Whole Genome Panel on an Ion Torrent PGM platform (Thermo Fisher Scientific, Waltham, MA, USA). Complete, high-quality sequences were obtained and sequencing performance was evaluated via four different metrics. As a subset of evaluated samples have been previously submitted for Sanger sequencing of the control region, a comparative analysis of both methods’ results was conducted in order to compare technique adequacy within a forensic context. Even though this study is one of the first to report full mtDNA genome sequences for Brazilian admixed populations, the observed haplotypes exhibit a predominance of Native American and African maternal lineages in the studied sample set, reproducing results described in the literature for control regions only. Interpopulation analysis among Brazilian and 26 worldwide populations was also carried out. The results indicate that MPS-generated full mtDNA genome sequences may have great utility in forensic real casework applications, with a pronounced gain of genetic information and discrimination power provided by coding region evaluation and the enhanced capacity of heteroplasmies determination. Database construction and other relevant factors concerning implementation of such techniques in Brazilian forensic laboratories are also discussed.

Abstract 1340: High NK cell number predicts poor overall survival in de novo treatment naive acute myeloid leukemia

Abstract Introduction Acute myeloid leukemia (AML) is a deadly disease associated with high mortality and morbidity. Previous studies report defects in NK cell maturation and function in AML patients leading to innate immune evasion. Considering that the number of NK cells may be an important indicator of innate immune regulation of AML, our group sought to understand the prognostic significance of NK cell number in de novo AML patients. Methods Patient samples were obtained from the Oregon Health and Science University (OHSU) Pathology Department between September 2010 and March 2016. This cohort study includes 82 newly diagnosed adult AML patients who had a bone marrow biopsy that was subjected to flow cytometry and a targeted-NGS panel within 30 days prior to induction treatment. Patients were excluded if they had antecedent hematological diseases or therapy-related AML. Bone marrow immunophenotyping was performed using the Ion Torrent PGM platform. Absolute numbers of NK cells were estimated from flow cytometric analysis of the bone marrow samples whereby lymphocytes were identified based on CD45 staining and light side scatter and NK cells were defined as CD3-/CD56+. Results This retrospective cohort of 82 newly diagnosed AML patients were 53.7% male with a median age of 61 years (range 18 - 83). Using 2017 ELN risk stratification guidelines, 36.6% of patients were classified as favorable, 26.8% as intermediate, and 36.6% as adverse. Absolute NK cell numbers in the bone marrow ranged from 1 to 896 cells/μl with a median of 98. When stratifying NK cells by staining intensity to identify subpopulations, the number of CD56 bright NK cells ranged from 0 to 69 cells/μl (median 3) and the number of CD56 dim NK cells ranged from 1 to 585 cells/μl (median 77). Overall survival (OS) was measured from diagnosis to date of death or last contact and varied from 1 day to 5.7 years (median 20.5 months). There were 3 deaths in the first week after diagnosis. Separate Cox proportional hazards regression models were applied to each NK cell group in both the univariable and multivariable settings, with the latter models accounting for patient age and ELN risk. Higher absolute numbers of NK cells (CD3-/CD56+) were significantly correlated with worse OS in both model settings, with a multivariable p-value of 0.001. The number of bright NK cells was a significant predictor of OS in the univariable model but lost significance when controlling for other variables. However, similar to the number of all NK cells and contrary to previous reports, higher counts of dim NK cells were significantly associated with shorter survival times, with a multivariable p-value of 0.001. Conclusions In this study, we show that higher NK cell numbers at diagnosis are associated with worse OS and thus play an important role in prognostication for AML patients. This association with survival is most evident in patients at least 60 years old or those with intermediate or adverse ELN risk. Citation Format: Weiwei Wang, Guang Fan, Andy Kaempf, evan Lind, Tomi Mori, Byung Park, Jennifer Saultz. High NK cell number predicts poor overall survival in de novo treatment naive acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1340.

Molecular versus morphological data for benthic diatoms biomonitoring in Northern Europe freshwater and consequences for ecological status

Diatoms are known to be efficient bioindicators for water quality assessment because of their rapid response to environmental pressures and their omnipresence in water bodies. The identification of benthic diatoms communities in the biofilm, coupled with quality indices such as the Indice de polluosensibilité spécifique (IPS) can be used for biomonitoring purposes in freshwater. However, the morphological identification and counting of diatoms species under the microscope is time-consuming and requires extensive expertise to deal with a constantly evolving taxonomy. In response, a molecular-based and potentially more cost-effective method has been developed, coupling high-throughput sequencing and DNA metabarcoding. The method has already been tested for water quality assessment with diatoms in Central Europe. In this study, we applied both the traditional and molecular methods on 180 biofilms samples from Northern Europe (rivers and lakes of Fennoscandia and Iceland). The DNA metabarcoding data were obtained on two different DNA markers, the 18S-V4 and rbcL barcodes, with the NucleoSpin Soil kit for DNA extraction and sequenced on an Ion Torrent PGM platform. We assessed the ability of the molecular method to produce species inventories, IPS scores and ecological status class comparable to the ones generated by the traditional morphology-based approach. The two methods generated correlated but significantly different IPS scores and ecological status assessment. The observed deviations are explained by presence/absence and abundance discrepancies in the species inventories, mainly due to the incompleteness of the barcodes reference databases, primer bias and strictness of the bioinformatic pipeline. Abundance discrepancies are less common than presence/absence discrepancies but have a greater effect on the ecological assessment. Missing species in the reference databases are mostly acidophilic benthic diatoms species, typical of the low pH waters of Northern Europe. The two different DNA markers also generated significantly different ecological status assessments. The use of the 18S-V4 marker generates more species inventories discrepancies, but achieves an ecological assessment more similar to the traditional morphology-based method. Further development of the metabarcoding method is needed for its use in environmental assessment. For its application in Northern Europe, completion and curation of reference databases are necessary, as well as evaluation of the currently available bioinformatics pipelines. New indices, fitted for environmental biomonitoring, should also be developed directly from molecular data.

Next-generation sequencing of BRCA1 and BRCA2 genes for rapid detection of germline mutations in hereditary breast/ovarian cancer.

BackgroundConventional methods used to identify BRCA1 and BRCA2 germline mutations in hereditary cancers, such as Sanger sequencing/multiplex ligation-dependent probe amplification (MLPA), are time-consuming and expensive, due to the large size of the genes. The recent introduction of next-generation sequencing (NGS) benchtop platforms offered a powerful alternative for mutation detection, dramatically improving the speed and the efficiency of DNA testing. Here we tested the performance of the Ion Torrent PGM platform with the Ion AmpliSeq BRCA1 and BRCA2 Panel in our clinical routine of breast/ovarian hereditary cancer syndrome assessment.MethodsWe first tested the NGS approach in a cohort of 11 patients (training set) who had previously undergone genetic diagnosis in our laboratory by conventional methods. Then, we applied the optimized pipeline to the consecutive cohort of 136 uncharacterized probands (validation set).ResultsBy minimal adjustments in the analytical pipeline of Torrent Suite Software we obtained a 100% concordance with Sanger results regarding the identification of single nucleotide alterations, insertions, and deletions with the exception of three large genomic rearrangements (LGRs) contained in the training set. The optimized pipeline applied to the validation set (VS), identified pathogenic and polymorphic variants, including a novel BRCA2 pathogenic variant at exon 3, 100% of which were confirmed by Sanger in their correct zygosity status. To identify LGRs, all negative samples of the VS were subjected to MLPA analysis.DiscussionOur experience strongly supports that the Ion Torrent PGM technology in BRCA1 and BRCA2 germline variant identification, combined with MLPA analysis, is highly sensitive, easy to use, faster, and cheaper than traditional (Sanger sequencing/MLPA) approaches.

Host and MTB genome encoded miRNA markers for diagnosis of tuberculosis

MicroRNAs (miRNAs) are a class of noncoding RNA molecules which are involved in various cellular and physiological processes. Previously, studies have identified several miRNAs that are potential diagnostic biomarkers for various infectious diseases including tuberculosis. We have performed small RNA sequencing using the Ion Torrent PGM platform in extra pulmonary tuberculosis (EPTB) subject's serum samples to identify circulating miRNAs during mycobacterium tuberculosis (MTB) infection. Our analysis identified 20 circulating miRNAs upregulated and 5 miRNAs downregulated during MTB infection in patient's serum. In addition, we have identified 6 MTB genome encoded miRNAs upregulated in EPTB patient's serum samples. Taqman based qRT-PCR analysis of host-genome encoded (hsa-miR-146a-5p and hsa-miR-125b-5p) and MTB genome encoded (MTB-miR5) miRNAs showed increased expression in a cohort of 52 healthy, pulmonary tuberculosis (PTB) and extra pulmonary tuberculosis (EPTB) patients serum samples. Our study identified for the first time a panel of host and MTB genome specific differentially expressed circulating miRNAs in serum samples of an Indian patient cohort with tuberculosis infection with a potential as a non-invasive diagnostic biomarker for tuberculosis infection.

Molecular diagnosis of somatic overgrowth conditions: A single-center experience.

BackgroundSomatic overgrowth conditions, including Proteus syndrome, Sturge–Weber syndrome, and PIK3CA‐related overgrowth spectrum, are caused by post‐zygotic pathogenic variants, result in segmental mosaicism, and give rise to neural, cutaneous and/or lipomatous overgrowth. These variants occur in growth‐promoting pathways leading to cellular proliferation and expansion of tissues that arise from the affected cellular lineage.MethodsWe report on 80 serial patients evaluated for somatic overgrowth conditions in a diagnostic laboratory setting, including three prenatal patients. In total, 166 tissues from these 80 patients were subjected to targeted sequencing of an 8‐gene panel capturing 10.2 kb of sequence containing known pathogenic variants associated with somatic overgrowth conditions. Deep next‐generation sequencing was performed with the IonTorrent PGM platform at an average depth typically >5,000×.ResultsLikely pathogenic or pathogenic variants were identified in 36 individuals and variants of unknown significance in four. The overall molecular diagnostic yield was 45% but was highly influenced by both submitted tissue type and phenotype. In the prenatal setting, two patients had pathogenic variants identified in cultured amniocytes but in a third patient, the pathogenic variant was only present in post‐natal tissues. Finally, expanding the test to include full gene sequencing of PIK3CA in contrast to targeted sequencing identified likely pathogenic variants in 3 of 7 patients that tested negative on the original panel.ConclusionNext‐generation sequencing has enabled sensitive detection of somatic pathogenic variants associated with overgrowth conditions. However, as the pathogenic variant allele frequency varies by tissue type within an individual, submission of affected tissue(s) greatly increases the chances of a molecular diagnosis.

Amplicon-Based Targeted Next-Generation Sequencing of Formalin-Fixed, Paraffin-Embedded Tissue.

Next-generation sequencing (NGS) is rapidly becoming the method of choice for mutation analysis in both research and diagnostics. The benefit of targeted NGS compared to whole-genome and whole-exome sequencing is that smaller amounts of input material can be used as well as qualitatively suboptimal tissue samples, like formalin-fixed, paraffin-embedded archival tissue.Here, we describe the protocol for targeted next-generation sequencing using the Ion Torrent PGM platform in combination with Ion Ampliseq NGS gene panels for formalin-fixed, paraffin-embedded tissues. Both the manual and the automated workflow are described as well as the bioinformatics for data analysis.

Analysis of Molecular Minimal Residual Disease in Patients with Acute Leukemia during Complete Remission

Introduction: Acute leukemia is a group of clonal heterogeneous diseases with high recurrence rate. The monitoring of minimal residual disease (MRD) after treatment mainly relies on flow cytometric immunophenotyping. Genetic abnormalities as the basis of the development of leukemia, the prognosis value of gene mutations in complete remission (CR) has yet to be established except for NPM1 and FLT3-ITD mutations. Recent studies have reported that persistent mutations in non-DNMT3A, TET2, and ASXL1 (DTA) mutations during CR had significant independent prognostic value with respect to relapse and survival rates, but the detection of persistent DTA mutations did not have such prognostic value (Jongen-Lavrencic M et al., N Eng J Med 2018). In this study, we analyzed the molecular MRD of mutations in 58 genes during CR in patients with acute leukemia and explored its impact on prognosis.Methods: Amplicon-targeted, next-generation sequencing (IonTorrent PGM platform, Thermo Fisher Scientific) of 58 genes that are frequently mutated in hematological malignancies was performed retrospectively on 202 patients of acute leukemia, including 117 cases of AML (median age, 23 years; rang, 1-65 years), 66 cases of B-ALL (median age, 13.5 years; rang, 1-45 years) and 19 cases of T-ALL (median age, 11 years; rang, 1-42 years). The average sequencing depth was 1000x. A minimum coverage of 100x and a variant allele frequency >5% were used as thresholds for calling variants.Results: Mutations presented in 8.5% (10/17) of AML patients during CR and the variant allele frequency (VAF) ranged from 5.0% to 25.5% (median VAF, 12%), of which 80% (8/10) were epigenetic regulator gene mutations (DNMT3A, TET2, and IDH1/2). DNMT3A mutation (R882 locus) were detected in five cases, IDH2 mutation (R140Q) were detected in two cases, IDH1 (R132C), TET2 (C1211Y), KIT (D816H), NPM1 (W288Cfs*12), and U2AF1 (S34F) mutation were each presented in one case. There were four cases with DTA and non-DTA mutations respectively and two cases concomitant with DTA and non-DTA mutations (one with DNMT3A and IDH1 mutations and another one with DNMT3A and NPM1 mutations). The median age of four patients with DTA mutations was 45 years (rang, 22-54 years), and the median follow-up time was 15 months (rang, 10-19 months). Among them, three cases were relapsed, two of the three patients who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT) were relapsed. The median age of four patients with non-DTA mutations was 24.5 years (rang, 12-64 years), and the median follow-up time was 25.5 months (rang, 12-35 months). All the patients with non-DTA mutations underwent allo-HSCT and were in continuous CR. One of the two patients who concomitant with DTA and non-DTA mutations (mutation in DNMT3A and IDH1) relapsed after allo-HSCT.Mutations presented in two cases of B-ALL patients during CR, of which PTPN11 (H520Y, VAF 5.9%) mutation was identified in one case who achieved MRD-negative after one course of induction and three courses of consolidation chemotherapy and in a state of sustained remission for two years after allo-HSCT at the time of reporting. KRAS (G12S, VAF 6.3%) mutation was identified in another case whose MRD persists positively after multiple courses of chemotherapy and relapsed in the third years after chemotherapy. At the time of reporting, the bone marrow is in a state of sustained remission for eight months after allo-HSCT. No mutations were detected in all T-ALL patient during CR.Conclusion: Mutations in the epigenetic regulators DNMT3A, TET2, and IDH1/2 are most common in patients with AML during complete remission and the majority of mutations in genes related to the RTK/RAS pathway were cleared. DTA mutations are more common in older patients. The recurrence rate after allo-HSCT is higher in patients with DTA mutation than that of patients with non-DTA mutation and which need to be further verified in the expanded cohort due to the limited number of cases in this study. In contrast to AML, mutations detected in B-ALL during complete remission were mainly RAS pathway and its prognostic significance needs to be further studied. DisclosuresNo relevant conflicts of interest to declare.

An alternative storage method for characterization of the intestinal microbiota through next generation sequencing.

ABSTRACTGut microbiota has been the subject of various molecular studies mainly due to its importance and wide-ranging relationships with human hosts. However, the storage of fecal samples prior to DNA extraction is critical when characterizing the composition of intestinal microbiota. Therefore, we aimed to understand the effects of different fecal storage methods to characterize intestinal microbiota using Next Generation Sequencing (NGS) as well as to establish an alternative conservation method of bacterial genetic material in these samples using guanidine. Stool samples from 10 healthy volunteers were collected. Each sample was divided into five aliquots: one aliquot was extracted immediately after collection (fresh) and two aliquots were subjected to freezing at -20 °C or -80 °C and extracted after 48 h. The other two aliquots were stored in guanidine at room temperature or 4 °C and extracted after 48 h. The V4 hypervariable regions of the bacterial and archeal 16S rRNA gene were amplified by PCR and sequenced using an Ion Torrent PGM platform for NGS. The data were analyzed using QIIME software. Statistical significance was determined using a non-parametric Kruskal-Wallis test. A total of 11,494,688 reads with acceptable quality were obtained. Unweighted principal coordinate analysis (PCoA) revealed that the samples were clustered based on the host rather than by the storage group. At the phylum and genus levels, we observed statistically significant differences between two genera, Proteobacteria (p=0.013) and Suterella (p=0.004), comparing frozen samples with guanidine-stored samples. Our data suggest that the use of guanidine can preserve bacterial genetic materials as well as freezing, providing additional conveniences.

Molecular analysis of inherited cardiomyopathy using next generation semiconductor sequencing technologies

BackgroundCardiomyopathies are the most common clinical and genetic heterogeneity cardiac diseases, and genetic contribution in particular plays a major role in patients with primary cardiomyopathies. The aim of this study is to investigate cases of inherited cardiomyopathy (IC) for potential disease-causing mutations in 64 genes reported to be associated with IC.MethodsA total of 110 independent cases or families diagnosed with various primary cardiomyopathies, including hypertrophic cardiomyopathy, dilated cardiomyopathy, restrictive cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, left ventricular non-compaction, and undefined cardiomyopathy, were collected after informed consent. A custom designed panel, including 64 genes, was screened using next generation sequencing on the Ion Torrent PGM platform. The best candidate disease-causing variants were verified by Sanger sequencing.ResultsA total of 78 variants in 73 patients were identified. After excluding the variants predicted to be benign and VUS, 26 pathogenic or likely pathogenic variants were verified in 26 probands (23.6%), including a homozygous variant in the SLC25A4 gene. Of these variants, 15 have been reported in the Human Gene Mutation Database or ClinVar database, while 11 are novel. The majority of variants were observed in the MYH7 (8/26) and MYBPC3 (6/26) gene. Titin (TTN) truncating mutations account for 13% in our dilated cardiomyopathy cases (3/23).ConclusionsThis study provides an overview of the genetic aberrations in this cohort of Chinese IC patients and demonstrates the power of next generation sequencing in IC. Genetic results can provide precise clinical diagnosis and guidance regarding medical care for some individuals.

Candidate gene based association analysis of salt tolerance in traditional and improved varieties of rice (Oryza sativa L.)

Rice is a staple food for more than half of the world population but the productivity of rice plant is attenuated in salt-stressed environment. Here 69 diverse rice varieties were evaluated for their salt tolerance capacity. Tolerant and susceptible genotypes were differentiated based on their standard evaluation score and thirteen other physiological parameters. A genome wide 48-SNP markers chip was used to analyze genetic diversity and population structure of the genotypes. Thirteen different salt-responsive candidate genes belonging to different functional categories including transporters, metabolic pathways, intermediate genes and transcription factors were amplified and re-sequenced using Ion Torrent PGM platform. Total 155 SNPs were identified in the selected 13 genes. A candidate gene based association analysis found seven-marker trait associations with five salt tolerance traits. From the 13 genes analyzed, DUF 6 was associated with Na+/K+ ratio, root biomass and root dry weight; SOS 1 with Na+ concentration in leaves and HKT 2;4 with shoot dry weight. This study will be helpful in the development of functional markers associated with salt stress tolerance in rice.

KPC-2 producing ST101 Klebsiella pneumoniae from bloodstream infection in India.

This study characterizes KPC-2 producing Klebsiella pneumoniae belonging to ST101. Whole genome sequencing using the Ion Torrent PGM platform with 400 bp chemistry was performed. blaKPC-2 was found on an IncFIIK plasmid associated with ISKpn6 and ISKpn7 without Tn4401. This is the first report of KPC-2 K. pneumoniae from bacteremia in India. The isolate also coded for other resistance genes such as aadA1, aadA2, armA, aac(3)-Ild, aac(6')-Ild for aminoglycoside; blaSHV-11, blaTEM-1B, blaOXA-9, for β-lactams and aac(6')-Ild, oqxA, oqxB, qnrB1 for fluoroquinolones. It belonged to the K17 capsular type. India is endemic to New Delhi metallo-β-lactamase and OXA48-like carbapenemases and K. pneumoniae carbapenemase (KPC) is seldom reported. With high rates of carbapenem resistance, emergence of KPC in India will challenge patient management. The isolate was susceptible to colistin. The patient had a fatal outcome.

Next-generation sequencing library preparation method for identification of RNA viruses on the Ion Torrent Sequencing Platform.

Next generation sequencing (NGS) is a powerful tool for the characterization, discovery, and molecular identification of RNA viruses. There were multiple NGS library preparation methods published for strand-specific RNA-seq, but some methods are not suitable for identifying and characterizing RNA viruses. In this study, we report a NGS library preparation method to identify RNA viruses using the Ion Torrent PGM platform. The NGS sequencing adapters were directly inserted into the sequencing library through reverse transcription and polymerase chain reaction, without fragmentation and ligation of nucleic acids. The results show that this method is simple to perform, able to identify multiple species of RNA viruses in clinical samples.

Abstract 504: MicroRNA Signatures in Murine Aorta and Carotid Arteries

Atherosclerosis, a major cause of mortality, affects arteries diffusely. However, some vascular beds are preferably involved. In the last decade microRNAs (miRs) have emerged as key regulators of gene expression in physiological or pathophysiological processes. Here, we investigated whether different arteries, commonly affected by atherosclerosis, present specific miR expression signatures. For this purpose, aorta (Ao) and carotid (Ca) arteries of four healthy male Wistar rats were dissected, total RNA was extracted with Trizol, and enriched for small RNA fraction. MicroRNAs were then sequenced using massive parallel sequencing (RNA-Seq) on Ion Torrent PGM platform. Data were analyzed using CLC Genomics Workbench software. We identified 266 mature miRs in Ao and 421 in Ca, 260 were common between arteries. Differential expression analysis (EDGE) showed increased expression (IE) of 16 miRs in Ao and reduced expression (RE) of 54 miRs in Ao, relative to Ca. The lists of differently expressed miRs were subjected to in silico functional analyzes: (1) computational target prediction, using miRWalk tool, identified 1,094 target genes for IE list and 3,574 for RE list; (2) gene-set enrichment analysis in the lists of putative target genes, using EnrichR tool, showed differences in enriched biological processes, such as response to shear stress, regulation of endothelial proliferation, smooth muscle metabolism, and response to FGF signaling (which were enriched in RE list); (3) construction of regulatory networks for differently expressed miRs, using miRnet, identified metabolic pathway components differently regulated between arteries, such as inhibition of SIRT1 and increased expression of HIF1-alpha in Ao. Taken together, our results show differences in miR signature between arteries, suggesting that specific expression profiles of miR may play a role in the vascular behavior as well as in atherosclerosis pathophysiology.