Ion Torrent PGM Research Articles

Abstract B50: Personna Onco: A multicancer panel for mutational screening in a Brazilian cohort

Abstract Next-generation sequencing (NGS) technology revolutionized the medical diagnosis field through concomitant analysis of multiple regions/genes and several individuals in a single experiment. This is currently allowing the transition from traditional to precision medicine, leading to a more accurate cancer diagnosis and enabling the best selection of molecular target drugs for individual treatments. In this context, the objective of this study was to survey the spectrum and prevalence of variants in solid tumor samples using Oncomine™ Focus Assay (Thermo Fisher Scientific), a multi-biomarker NGS assay for the detection of relevant alterations (hotspots, SNVs, indels, CNVs, and gene fusions) in 52 oncogenes associated with different solid tumors. Formalin-fixed, paraffin-embedded (FFPE) samples were obtained from 362 patients (186 males and 176 females) and clinical-pathologic data were obtained from pathologist/physician responsible for each patient. Genomic DNA and total RNA were extracted from all samples. Libraries were prepared and sequenced on Ion Torrent PGM sequencer. Sequencing data was analyzed using Ion Reporter software integrated with Oncomine® Knowledgebase. A total of 106 patients (29.3%) did not present any variants and only 13 patients (3.6%) showed inconclusive results due to poor DNA/RNA quality and/or quantity. These patients were excluded from further analysis. In general, 68 CNVs, 36 fusion genes, and 245 point mutations/indels were observed for the remaining patients (n=243). The most frequent tumor type observed was lung cancer (46.5%) showing EGFR (34.5%) and KRAS (31%) as the most frequently altered genes. These mutations are: KRAS G12 hotspot (22.1%: p.G12C - 8.0%, p.G12D - 7.1%, p.G12V - 4.4%, p.G12A - 1.8%, p.G12F – 0.9%), EGFR p.E746_A750del (6.2%), and EGFR p.L858R (6.2%). Other observed tumors were colon, breast, bladder, ovary, liver, uterus, brain, skin, and bone. Interestingly, this panel allowed the identification of not commonly observed variants in these tumors such as ovary adenocarcinoma PIK3CA p.M1043I, moderately differentiated colon adenocarcinoma FGFR4 amplification, lung adenocarcinoma BRAF p.G469A, and ERBB2 amplification. All these alterations were previously associated with drug response for other cancer types. This study demonstrated that a multicancer panel plays an important role for clinicians and patients, allowing the identification of multiple druggable variants with approved therapies, potentially improving patient outcomes, and increasing the chances of getting the best treatment. In addition, the generation of large-scale results of variants in tumors contributes to the description of mutations classified as nonpathogenic and that may eventually become driver mutations. Citation Format: Maíra Cristina Menezes Freire, Michele Araújo Pereira, Priscila Daniele Ramos Cirillo, Natália Penido Lopes, Carolina Bustamante, Rafael Lucas Muniz Guedes, Mariano Zalis. Personna Onco: A multicancer panel for mutational screening in a Brazilian cohort [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B50.

Abstract A30: Depicting clinically relevant molecular alterations in oropharynx squamous cell carcinoma samples treated with an organ preservation protocol

Abstract Introduction: Head and neck cancers are among the most common human malignancies in the world, with high rates of morbidity and mortality. These tumors are mostly caused by heavy tobacco smoking; however, high-risk human papillomavirus (HR-HPV) infection has been increasingly associated with tumors in the oropharynx. Oropharynx squamous cell carcinomas (OPSCC) associated with HR-HPV infection represent a distinct clinical and epidemiologic entity, with differences in clinical presentation and treatment outcome reflecting distinct genetic and biologic profiles. Initial treatment decisions are critical to improve patient survival; therefore, considering that multimodality therapy is often toxic, treatment choice should balance tolerability, function preservation, and global response to therapy. Molecular markers may be beneficial in a better classification of patients, thus aiding in a more consistent tailoring of patient treatment and follow-up. Objective: The aim of this study was to identify specific clinical and molecular profiles that classify subgroups of OPSCC patients according to their outcome. Materials and Methods: Thirty OPSCC samples from patients treated with a chemoradiation-based organ-preservation protocol were included in this study. P16 protein expression was used as a surrogate marker for HR-HPV. The methylation status of 5 genes was evaluated by quantitative methylation-specific PCR (qMSP), and next-generation sequencing of a 15-gene panel was performed using the Ion Torrent PGM platform. Results: The majority of patients (93.3%) were male, tobacco users (85.2%), presented with advanced disease (90%), and 36.7% were HR-HPV positive. Specific gene-promoter hypermethylation was observed in 32.1-50% of the OPSCC evaluated and, as expected, CCNA1 methylation was associated with HPV(+ve) cases (p=0.035). Eighteen patients (58.1%) had at least 1 gene mutated: 87.5% of HPV(-ve) and 57.1% of HPV(+ve) cases. Interestingly, 60% of smokers HPV(+ve) cases were wild-type for TP53 variants (p=0.003). The absence of deleterious variants in several genes (EZH2, FAT1, FBWX7, HRAS, IRF6, PIK3CA, and PTEN) was significantly associated with a complete response of the primary tumor site to the organ-preservation treatment protocol (p<0.004). Conclusions: Even though all samples analyzed were of the oropharynx, several differences regarding molecular and clinical characteristics were identified. An association between molecular markers and the profile of therapy response can be useful in decision-making of the initial treatment and improve patient survival. More cases are being evaluated in order to assess whether the differences identified are able to significantly classify these patients into molecular subgroups associated with different outcomes and help in more thorough patient management. Citation Format: Ana Carolina de Carvalho, Matias Eliseo Melendez, Cristovam Scapulatempo Neto, Gabriela Carvalho Fernandes, Lidia Maria Rebolho Batista Arantes, Bruna Pereira Sorroche, Gisele Caravina de Almeida, Rui Manuel Reis, Sandra Perdomo, André Lopes Carvalho. Depicting clinically relevant molecular alterations in oropharynx squamous cell carcinoma samples treated with an organ preservation protocol [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A30.

Impact of clay minerals on bacterial diversity during the fermentation process of kimchi

Abstract Kimchi is a popular traditional Korean food and has various beneficial health properties, e.g., it has preventative effects against cancer, obesity, diabetes, and constipation. The characteristics of kimchi, including its texture, flavor, and functionality, are affected by the major and minor ingredients. Bentonite, a clay mineral, is used as a component in functional foods, cosmetics, and pharmaceuticals owing to its physical and chemical characteristics. In this study, different types of cation-substituted forms of bentonite (Na+, K+ and Mg2 +) were used as kimchi ingredients to analyze their effect on the microbial community during fermentation for 10 days. Amplicon sequencing targeting the bacterial 16S rRNA gene was conducted using the ion torrent PGM system and sequencing data were analyzed with Qiime. At the initial stage, kimchi samples exhibited diverse microflora. Lactic acid bacteria (LAB), such as Weissella, Lactobacillus, and Lactococcus, dominated after 10 days of fermentation. Seven core LAB species, i.e., Leuconostoc gelidum, Leuconostoc gasicomitatum, Weissella koreensis, Leuconostoc citreum, Leuconostoc lactis, Lactobacillus sakei, and Weissella cibaria, showed differences in abundance among samples treated with different types of bentonite.

Assessment of bacterial communities and activities of thermotolerant enzymes produced by bacteria indigenous to oil-bearing sandstone cores for potential application in Enhanced Oil Recovery

Enhanced Oil Recovery (EOR) is a potential approach to improve oil yield in petroleum production. In this research, indigenous bacterial communities residing in oil-bearing sandstone cores, taken from oil wells in Fang oil field in Northern Thailand, were investigated using the Ion Torrent PGM sequencing method. Based on 16rRNA gene sequences, Proteobacteria and Firmicutes were found to be the predominant phyla, and Bacillus, Sinomonas, Paenibacillus and Hydrogenophaga were the major genera. Thermotolerant lipase-esterase and urease producing bacteria were also isolated from the oil-bearing sandstone core samples. One lipase-esterase producing isolate, Bacillus licheniformis L3-2, produced the enzyme with highest activity at 80 °C. The enzyme retained up to 50% of its activity after incubation at 60 °C for 4 h. The results suggest the possibility of applying an in situ EOR approach using a combination of Enzyme Enhanced Oil Recovery (EEOR) and Microbial Enhanced Oil Recovery (MEOR) methods, with this lipase-esterase producing Bacillus licheniformis isolate, which is indigenous to one of the wells in this oil field.

Establishing the Ion Torrent PGM Sequencing Methods for the Clinical Diagnosis of MPN Patients

To investigate the feasibility and relibility of rapidly and accurately acquiring the informations of gene mutations in MPN patients by using self-designed custom MPN mutation-related multipe-PCR primer kit and next generation Ion Torrent PGM sequencing platform. The bone marrow samples of 10 MPN patients with JAK2V617F and/or CALR+, Ph- confirmed by sanger sequencing method were collected and were re-detected by using next generation Ion Torrent PGM sequencing method, then the consistence of results of above-mentioned 2 kinds of detection methods was compared. In terms of JAK2V617F, MPL and CALR mutations, the results of Ion Torrent PGM sequencing were complete consistent with results of Sanger sequencing, except 52 bp deletion of CALR gene, which conld not be detected by next generation Ion Torrent PGM sequencing method in all bone marrow samples. The detection of multiple gene mutations in MPN patients by Ion Torrent PGM sequencing platform is feasible and can meet the needs of clinical testing. This method can complete detection of all 23 mutetions within 1-2 days, moreover, possesses advantages of high sensitivity, specificity, rapidity, high throughput and low cost.

HLA genotyping by next-generation sequencing of complementary DNA

BackgroundGenotyping of the human leucocyte antigen (HLA) is indispensable for various medical treatments. However, unambiguous genotyping is technically challenging due to high polymorphism of the corresponding genomic region. Next-generation sequencing is changing the landscape of genotyping. In addition to high throughput of data, its additional advantage is that DNA templates are derived from single molecules, which is a strong merit for the phasing problem. Although most currently developed technologies use genomic DNA, use of cDNA could enable genotyping with reduced costs in data production and analysis. We thus developed an HLA genotyping system based on next-generation sequencing of cDNA.MethodsEach HLA gene was divided into 3 or 4 target regions subjected to PCR amplification and subsequent sequencing with Ion Torrent PGM. The sequence data were then subjected to an automated analysis. The principle of the analysis was to construct candidate sequences generated from all possible combinations of variable bases and arrange them in decreasing order of the number of reads. Upon collecting candidate sequences from all target regions, 2 haplotypes were usually assigned. Cases not assigned 2 haplotypes were forwarded to 4 additional processes: selection of candidate sequences applying more stringent criteria, removal of artificial haplotypes, selection of candidate sequences with a relaxed threshold for sequence matching, and countermeasure for incomplete sequences in the HLA database.ResultsThe genotyping system was evaluated using 30 samples; the overall accuracy was 97.0% at the field 3 level and 98.3% at the G group level. With one sample, genotyping of DPB1 was not completed due to short read size. We then developed a method for complete sequencing of individual molecules of the DPB1 gene, using the molecular barcode technology.ConclusionThe performance of the automatic genotyping system was comparable to that of systems developed in previous studies. Thus, next-generation sequencing of cDNA is a viable option for HLA genotyping.

Preliminary study of exon sequence in pneumoconiosis and pneumoconiosis complicated with lung cancer using high-throughput sequencing technology

Objective: The detection and analysis of exon mutations of pneumoconiosis and pneumoco-niosis complicated with lung cancer provide reference evidence for screening, clinical diagnosis and treatment and prognosis in pneumoconiosis and pneumoconiosis complicated with lung cancer. Methods: The pathologi-cal tissue samples from 3 pneumoconiosis patients and 3 pneumoconiosis complicated with lung cancer pa-tients were collected. Genomic DNA was extracted and library was prepared. Exomes of the pathological tissue samples in pneumoconiosis patients and pneumoconiosis complicated with lung cancer patients were se-quenced using Ion Torrent PGM platform. Results: Mutation genes FGFR3, PDGFRA, KDR, APC, EGFR, FGFR2, SMO, TP53, RET and CDKN2A were detected in pathological tissue samples of 3 pneumoconiosis patients; Mutation genes FGFR3, PDGFRA, KIT, KDR, APC, EGFR, FGFR2, TP53, RET, CDKN2A, ATM, NPM1, MET and FLT3 were detected in pathological tissue samples of 3 pneumoconiosis complicated with lung cancer patients (P<0.01) . FGFR3, PDGFRA were detected in pathological tissue samples of each pa-tient (mutation frequency>98%) ; Differential genes KIT, ATM, NPM1, MET and FLT3 were only detected in pathological tissue samples of pneumoconiosis complicated with lung cancer patients but not pneumoconio-sis patients. Conclusion: A variety of exon mutations detected in pneumoconiosis patients and pneumoconiosis complicated with lung cancer patients using high-throughput sequencing technique have potential value of ap-plications in screening, clinical diagnosis and treatment and prognosis in pneumoconiosis and pneumoconiosis complicated with lung cancer.

16S rRNA gene sequencing on a benchtop sequencer: accuracy for identification of clinically important bacteria.

AimsTest the choice of 16S rRNA gene amplicon and data analysis method on the accuracy of identification of clinically important bacteria utilizing a benchtop sequencer.Methods and ResultsNine 16S rRNA amplicons were tested on an Ion Torrent PGM to identify 41 strains of clinical importance. The V1–V2 region identified 40 of 41 isolates to the species level. Three data analysis methods were tested, finding that the Ribosomal Database Project's SequenceMatch outperformed BLAST and the Ion Reporter Metagenomics analysis pipeline. Lastly, 16S rRNA gene sequencing mixtures of four species through a six log range of dilution showed species were identifiable even when present as 0·1% of the mixture.ConclusionsSequencing the V1–V2 16S rRNA gene region, made possible by the increased read length Ion Torrent PGM sequencer's 400 base pair chemistry, may be a better choice over other commonly used regions for identifying clinically important bacteria. In addition, the SequenceMatch algorithm, freely available from the Ribosomal Database Project, is a good choice for matching filtered reads to organisms. Lastly, 16S rRNA gene sequencing's sensitivity to the presence of a bacterial species at 0·1% of a mixture suggests it has sufficient sensitivity for samples in which important bacteria may be rare.Significance and Impact of the StudyWe have validated 16S rRNA gene sequencing on a benchtop sequencer including simple mixtures of organisms; however, our results highlight deficits for clinical application in place of current identification methods.

488O - Serum miRNA discriminates treatment-naive localized synovial sarcoma patients from those in follow-up after radical combined therapy

Background: Synovial sarcoma (SySa) is an aggressive neoplasm that accounts for approximately 10% of all soft-tissue tumors. The levels of miRNAs in blood circulation was investigated in the past for SySa patients with array technology, however to date no exploratory next-generation sequencing (NGS) approach was used to evaluate blood circulating miRNA as response markers of SySa treatment. We present data coming from patients with locally advanced synovial sarcoma treated according to a uniform protocol of preoperative therapy (chemotherapy and radiotherapy). Methods: Serum samples of 28 SySa patients prior to preoperative therapy, following two courses of neoadjuvant chemotherapy (n = 23) and in follow-up after radical radiotherapy and surgery (n = 18) as well as the serum of healthy 30 donors were collected. MiRNAs were isolated from serum with MirVANA miRNA Isolation Kit and then analyzed using deep sequencing on Ion Torrent PGM. Reads were mapped to miRBase miRNA collection with miRDeep2. Differential expression was evaluated with edgeR. Comparisons were made between samples before treatment and those during treatment, in observation after treatment as well as healthy controls. Separate comparisons were also made for samples before and after surgical treatment. Results: Deep sequencing identified 1370 miRNAs. 47 miRNAs differentiated (adj. p- value <0.05) samples after treatment or in observation from those before chemotherapy and/or surgery. Out of these, two miRNAs were present in both comparisons and had good discriminative properties: hsa-miR-31-5p and hsa-miR-127-5p (Area Under the Curve - AUCs ranging from 0.75 to 0.87). In addition 49 miRNAs differentiated patients before treatment from healthy controls, 8 of which were also present in previous comparisons (hsa-miR-423-5p, hsa-miR-320a, hsa-miR-339-5p, hsa-miR-214-3p, hsa-miR-486-3p, hsa-miR-150-5p, hsa-miR-214-5p, hsa-miR-769-5p). However, none of these miRNAs reached AUCs above 0.8. Conclusions: Our NGS-based approach have identified blood circulating miRNAs with high to good discriminative properties to differentiate treatment-naive and SySa patients after treatment or in observation following the surgery. Further evaluation on an independent group of patients is warranted to confirm their value as a biomarker for the diagnosis of SySa recurrence or metastasis. Legal entity responsible for the study: Piotr Rutkowski Funding: National Science Center [2013/11/B/NZ5/03165 to PR] Disclosure: All authors have declared no conflicts of interest.

A novel mutation of the EYA4 gene associated with post-lingual hearing loss in a proband is co-segregating with a novel PAX3 mutation in two congenitally deaf family members

ObjectivesThis work was aimed at establishing the molecular etiology of hearing loss in a 9-year old girl with post-lingual non-syndromic mild sensorineural hearing loss with a complex family history of clinically heterogeneous deafness. MethodsThe proband's DNA was subjected to NGS analysis of a 59-targeted gene panel, with the use of the Ion Torrent PGM platform. Conventional Sanger sequencing was used for segregation analysis in all the affected relatives. The proband and all the other hearing impaired members of the family underwent a thorough clinical and audiological evaluation. ResultsA new likely pathogenic mutation in the EYA4 gene (c.1154C > T; p.Ser385Leu) was identified in the proband and in her 42-year-old father with post-lingual non-syndromic profound sensorineural hearing loss. The EYA4 mutation was also found in the proband's grandfather and uncle, both showing clinical features of Waardenburg syndrome type 1. A novel pathogenic splice-site mutation (c.321+1G > A) of the PAX3 gene was found to co-segregate with the EYA4 mutation in these two subjects. ConclusionThe identified novel EYA4 mutation can be considered responsible of the hearing loss observed in the proband and her father, while a dual molecular diagnosis was reached in the relatives co-segregating the EYA4 and the PAX3 mutations. In these two subjects the DFNA10 phenotype was masked by Waardenburg syndrome.The use of NGS targeted gene-panel, in combination with an extensive clinical and audiological examination led us to identify the genetic cause of the hearing loss in members of a family in which different forms of autosomal dominant deafness segregate. These results provide precise and especially important prognostic and follow-up information for the future audiologic management in the youngest affected member.

Mitochondrial recovery from shotgun metagenome sequencing enabling phylogenetic analysis of the common thresher shark (Alopias vulpinus)

The common thresher shark (Alopias vulpinus) is an important fisheries species in the northeastern Pacific Ocean along California, USA and Mexico; yet genetic understanding of this species is incomplete. Using IonTorrent PGM generated metagenomic libraries constructed from the skin surface, we recovered the complete mitogenome of the common thresher shark. The length of the mitogenome was 16,712 basepairs and consisted of 22 tRNA, two rRNA, 13 protein coding sequences, a replication origin and a control region, similar to other Alopias spp. The median coverage across the mitogenome was 21×, ranging from 1 to 48× coverage. The mean GC content of the mitogenome was 37.83%. Using the 13 protein coding genes of the mitogenome, we show phylogenetic placement of the common thresher shark with the pelagic thresher (Alopias pelagicus; a posterior probability of 1). In addition, the inclusion of this mitogenome increased certainty for the placement of the Lamniformes family with the Carcharhiniformes family (a posterior probability of 1), rather than with the Orectolobeformes family, as has been previously reported. The availability of the common thresher shark mitogenome will aid phylogenetic inference and population based studies of this important fisheries species.

Genomic GC-Content Affects the Accuracy of 16S rRNA Gene Sequencing Based Microbial Profiling due to PCR Bias.

Profiling of microbial community composition is frequently performed by partial 16S rRNA gene sequencing on benchtop platforms following PCR amplification of specific hypervariable regions within this gene. Accuracy and reproducibility of this strategy are two key parameters to consider, which may be influenced during all processes from sample collection and storage, through DNA extraction and PCR based library preparation to the final sequencing. In order to evaluate both the reproducibility and accuracy of 16S rRNA gene based microbial profiling using the Ion Torrent PGM platform, we prepared libraries and performed sequencing of a well-defined and validated 20-member bacterial DNA mock community on five separate occasions and compared results with the expected even distribution. In general the applied method had a median coefficient of variance of 11.8% (range 5.5–73.7%) for all 20 included strains in the mock community across five separate sequencing runs, with underrepresented strains generally showing the largest degree of variation. In terms of accuracy, mock community species belonging to Proteobacteria were underestimated, whereas those belonging to Firmicutes were mostly overestimated. This could be explained partly by premature read truncation, but to larger degree their genomic GC-content, which correlated negatively with the observed relative abundances, suggesting a PCR bias against GC-rich species during library preparation. Increasing the initial denaturation time during the PCR amplification from 30 to 120 s resulted in an increased average relative abundance of the three mock community members with the highest genomic GC%, but did not significantly change the overall evenness of the community distribution. Therefore, efforts should be made to optimize the PCR conditions prior to sequencing in order to maximize accuracy.

Indigenous species barcode database improves the identification of zooplankton.

Incompleteness and inaccuracy of DNA barcode databases is considered an important hindrance to the use of metabarcoding in biodiversity analysis of zooplankton at the species-level. Species barcoding by Sanger sequencing is inefficient for organisms with small body sizes, such as zooplankton. Here mitochondrial cytochrome c oxidase I (COI) fragment barcodes from 910 freshwater zooplankton specimens (87 morphospecies) were recovered by a high-throughput sequencing platform, Ion Torrent PGM. Intraspecific divergence of most zooplanktons was < 5%, except Branchionus leydign (Rotifer, 14.3%), Trichocerca elongate (Rotifer, 11.5%), Lecane bulla (Rotifer, 15.9%), Synchaeta oblonga (Rotifer, 5.95%) and Schmackeria forbesi (Copepod, 6.5%). Metabarcoding data of 28 environmental samples from Lake Tai were annotated by both an indigenous database and NCBI Genbank database. The indigenous database improved the taxonomic assignment of metabarcoding of zooplankton. Most zooplankton (81%) with barcode sequences in the indigenous database were identified by metabarcoding monitoring. Furthermore, the frequency and distribution of zooplankton were also consistent between metabarcoding and morphology identification. Overall, the indigenous database improved the taxonomic assignment of zooplankton.

A targeted next-generation sequencing panel reveals novel mutations in Japanese patients with primary ciliary dyskinesia

ObjectivePrimary ciliary dyskinesia (PCD) is a rare genetic disorder caused by functional impairment of cilia throughout the body. The early diagnosis of PCD is important for the prevention of long-term sequelae; however, this is often challenging because of the phenotypic heterogeneity of PCD and difficulty in genetic analysis. The majority of PCD patients in Japan are not diagnosed properly. To diagnose PCD more accurately, we developed a targeted next-generation sequencing (NGS) panel. MethodsWe examined 46 patients (age range, 1–64 years; 23 male and 23 female) who were clinically suspected of PCD. First, mutation hotspots in DNAH5 and DNAI1 were sequenced by the Sanger method. Next, exome sequencing was performed in 32 known PCD genes using our novel NGS panel with the Ion Torrent PGM system. Variant annotation was generated by Ion Reporter Version 5.0 (Life Technologies). Mutations found in the panel were validated by Sanger sequencing. ResultsDisease-causing gene mutations were found in 10 patients from 7 families: DNAH5 in 4 families, and DNAI1, CCDC40, and RSPH4A in 1 family each. Heterozygous mutations were found in 1 patient. The majority of the mutations found in the present analysis were novel. ConclusionJapanese PCD patients have novel mutations in cilia-related genes. This targeted NGS panel can identify disease-causing mutations in patients with PCD.

Complete genome sequence of a novel lytic bacteriophage isolated from Ralstonia solanacearum.

A lytic podophage RSPI1 was isolated from tobacco field soil collected in Fujian Province, South China using host bacterium Ralstonia solanacearum Tb15-14. Whole genome sequencing of this phage was performed using the high-throughput Ion Torrent PGM Sequencer. The complete genome of RSPI1 was 43,211 bp in length with a mean DNA G + C content of 61.5%. A total of 48 open reading frames were identified with lengths ranging from 132 bp to 5,061 bp, of which, 11, 12 and 25 were identified as functional, structural and unknown genes, respectively. A BLAST analysis revealed that this phage genome had a query cover of 78-79% and a highest identity of 84% with four podophages that infect Burkholderia pseudomallei. Two neighbor-joining phylogenetic trees were constructed using phage DNA polymerase I and tail fiber protein sequences and showed that this phage is closely related to Burkholderia phage Bp-AMP1, and also related to several phages that infect Ralstonia solanacearum. These findings indicate that RSPI1 is a novel phage that infects the notorious plant pathogen Ralstonia solanacearum.

A comparison of sequencing platforms and bioinformatics pipelines for compositional analysis of the gut microbiome

BackgroundAdvancements in Next Generation Sequencing (NGS) technologies regarding throughput, read length and accuracy had a major impact on microbiome research by significantly improving 16S rRNA amplicon sequencing. As rapid improvements in sequencing platforms and new data analysis pipelines are introduced, it is essential to evaluate their capabilities in specific applications. The aim of this study was to assess whether the same project-specific biological conclusions regarding microbiome composition could be reached using different sequencing platforms and bioinformatics pipelines.ResultsChicken cecum microbiome was analyzed by 16S rRNA amplicon sequencing using Illumina MiSeq, Ion Torrent PGM, and Roche 454 GS FLX Titanium platforms, with standard and modified protocols for library preparation. We labeled the bioinformatics pipelines included in our analysis QIIME1 and QIIME2 (de novo OTU picking [not to be confused with QIIME version 2 commonly referred to as QIIME2]), QIIME3 and QIIME4 (open reference OTU picking), UPARSE1 and UPARSE2 (each pair differs only in the use of chimera depletion methods), and DADA2 (for Illumina data only). GS FLX+ yielded the longest reads and highest quality scores, while MiSeq generated the largest number of reads after quality filtering. Declines in quality scores were observed starting at bases 150–199 for GS FLX+ and bases 90–99 for MiSeq. Scores were stable for PGM-generated data. Overall microbiome compositional profiles were comparable between platforms; however, average relative abundance of specific taxa varied depending on sequencing platform, library preparation method, and bioinformatics analysis. Specifically, QIIME with de novo OTU picking yielded the highest number of unique species and alpha diversity was reduced with UPARSE and DADA2 compared to QIIME.ConclusionsThe three platforms compared in this study were capable of discriminating samples by treatment, despite differences in diversity and abundance, leading to similar biological conclusions. Our results demonstrate that while there were differences in depth of coverage and phylogenetic diversity, all workflows revealed comparable treatment effects on microbial diversity. To increase reproducibility and reliability and to retain consistency between similar studies, it is important to consider the impact on data quality and relative abundance of taxa when selecting NGS platforms and analysis tools for microbiome studies.

1514P - Serum miRNA abundances discriminate imatinib-naive patients with advanced gastrointestinal stromal tumors (GIST) from those in remission on Imatinib therapy

Background: Deregulation of microRNAs (miRNAs) expression is observed virtually in all major types of neoplasm and miRNAs level in blood circulation are investigated as a potential diagnostics or prognostics biomarkers for neoplastic disorders. Gastrointestinal stromal tumors (GISTs) is the most common sarcoma of the gastrointestinal tract and to date performed studies on GISTs have provided mounting evidence on altered miRNA association with clinical, pathological features and Imatinib resistance in GIST. However, the utility of circulating miRNA as response markers of GIST progression and for Imatinib treatment have not been evaluated Methods: 36 metastatic or unresectable CD-117-positive GIST patients, were enrolled and serum sample was collected prior to Imatinib treatment. All patients responded initially to imatinib therapy. In 12 patients an additional serum sample was collected following targeted treatment at the time of remission. Control group comprised 30 healthy individuals. MiRNAs were isolated from serum with MirVANA miRNA Isolation Kit and then analyzed using deep sequencing on Ion Torrent PGM. Reads were mapped to miRBase miRNA collection with miRDeep2. Differential expression was evaluated with edgeR. Results: Deep sequencing identified 1284 miRNAs. The pair-wise comparison between Imatinib treated and Imatinib-naive GIST samples uncovered 22 miRNAs with differential expression (adjusted p value <0.05) of which 10 (miR-142-5p, let-7d-3p, miR-30e-5p, miR-194-5p, miR-223-5p, miR-223-3p, miR-125a-5p, miR-199b-5p, miR-24-2-5p, miR-641) yielded AUCs (areas under Receiver Operating Characteristic curves) ranging 0.81 and 0.9, thus having a high discriminative properties. A comparison of imatinib-naive GIST and control healthy samples revealed 99 differentially expressed miRNAs (adjusted p value <0.05) of which four (miR-582-5p, miR-150-5p, miR-450b-5p, miR-450a-5p) reached AUCs with high discriminatory power ranging 0.81-0.84. Conclusions: Circulating miRNA abundances can distinguish GIST patients from those in remission following Imatinib therapy as well as from the healthy controls. However, further studies evaluating the potential of designated microRNAs as response markers for treatment or as predictive markers of GIST are warranted. Legal entity responsible for the study: Piotr Rutkowski Funding: the grant from National Science Center [2013/11/B/NZ5/03165 to PR Disclosure: All authors have declared no conflicts of interest.

Genomic characterization of Zika virus isolated from Indonesia

Zika virus (ZIKV) JMB-185 strain was isolated from a febrile patient in Jambi, Indonesia in 2014. To understand its genetic characteristics, we performed whole genome sequencing using the Ion Torrent PGM platform on the supernatant of the first passage. The phylogenetic analysis showed that the isolate was not closely related to the Brazilian ZIKV associated with microcephaly or isolates from the recent Singapore Zika outbreak. Molecular evolution analysis indicated that JMB-185 strain may have been circulating in the Southeast Asia region, including Indonesia since 2000. We observed high nucleotide sequence identity between Indonesia, Thailand, Singapore, and American strains although unique amino acid substitutions were also observed. This report provides information on the genomic characteristics of Indonesian ZIKV which may be used for further studies.

Abstract 3945: Precise copy-number profiling of single cells isolated from FFPE tissues by low-pass whole-genome sequencing

Abstract Introduction Chromosome instability (CIN) is a hallmark of cancer, acting by boosting genetic alterations responsible of tumorigenesis, progression and heterogeneity. Whole-genome sequencing (WGS) protocols are established methods for studying copy-number alterations (CNA) in single-cells, following a necessary whole-genome amplification (WGA) step. We previously presented a method for single-cell CNA profiling of CTCs based on shallow WGS of LM-PCR based WGA products. Here for the first time, we show that the same method may be employed even on single FFPE cells, overcoming the challenges of DNA degradation and damage linked to this type of samples. Methods Two 50μm thick FFPE sections from pancreas ductal adenocarcinoma with &amp;lt;20% tumor content were processed following DEPArray™ sample preparation protocol, including cell dissociation and staining with vimentin, keratin and DAPI, and DNA quality was assessed using the DEPArray FFPE QC kit (QC=0.7). Cell pools (n=15, range=6-213 cells) and single-cells (n=11) from pure tumor and stromal populations were recovered in separate tubes and lysed. A pool of 86 tumor cells and a pool of 213 stromal cells were used for low-pass WGS on MiSeq, obtaining ≈2M paired-end reads on average. By contrast, other cell pools and single cells were firstly whole-genome amplified using the Ampli1™ WGA kit and then sequenced at shallow coverage using Ampli1 LowPass kit and IonTorrent PGM, with ≈500k single-end reads as mean throughput per library. The genome integrity (GI) of WGA samples was evaluated by Ampli1 QC, a PCR-assay based on 4 amplicons of different lengths. Results The purity of DEPArray sorting was confirmed by the large number of chromosome alterations in sorted tumor and the parallel lack of gains and losses in sorted stromal pools and single-cells. A titration test was conducted measuring the consistency of CNA profiles of tumor WGA products starting from different number of cells, ranging from 117 down to single cells. For the purpose, we employed a Receiver Operating Characteristic (ROC) curve using the non-amplified tumor population as reference. Results showed an excellent performance level with a mean Area Under Curve (AUC) equals to 0.93. Interestingly, lower AUCs (0.87) were observed for single-cells, due to some level of inter-cell heterogeneity. Moreover, the proposed low-pass WGS method demonstrated a high resiliency to DNA degradation as quality of CNA profiles, measured by Derivative Log Ratio Spread (DLRS), only showed a weak correlation with GI level, with high-quality CNA profiles obtained also with the lowest GI value. Conclusions Presented approach for copy-number profiling of tumor single-cells isolated by DEPArray digital sorter and processed with Ampli1 workflow has proven to be a reliable and valuable application for the molecular characterization of tumor clones in degraded samples as FFPE tissues. Citation Format: Alberto Ferrarini, Genny Buson, Chiara Bolognesi, Claudio Forcato, Paola Tononi, Valentina del Monaco, Chiara Mangano, Francesca Fontana, Gianni Medoro, Nicolò Manaresi. Precise copy-number profiling of single cells isolated from FFPE tissues by low-pass whole-genome sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3945. doi:10.1158/1538-7445.AM2017-3945

Evolution and diversity studies of innate immune genes in Indian buffalo (Bubalus bubalis) breeds using next generation sequencing

Indian buffalo (Bubalus bubalis) breeds are acknowledged for their disease resistance power, hardiness and considerable milk production. River buffaloes are more disease resistance despite of tropical environment of India than cattle breeds of the world. The genetic polymorphisms of innate immune genes play a prominent role in disease susceptibility of livestock. Here in our study, we mainly focused on SNPs in ten selected candidate innate immune genes viz CHGA, CHGB, CHGC, Slc11a1, Slc11a2, DEFB1, BNBD4, BNBD5, LAP and TAP among three most economically important Indian buffalo breeds viz., Jaffrabadi, Mehsani and Murrah using amplicon sequencing on Ion torrent PGM sequencing platform. Polymorphism identification using by GATK tool of Unified Genotyper revealed 274, 245 and 224 high quality SNPs in Jaffrabadi, Mehsani and Murrah buffalo breeds, respectively as well as 168 common SNPs among these three breeds and uniquely detected SNPs were 31, 15 and 37 SNPs in Jaffrabadi, Mehsani and Murrah breeds respectively. Consequences of non-synonymous SNPs were analysed using several computational tools viz., I-Mutant, SIFT, PolyPhen and PROVEAN which identified three deleterious nsSNPs. This wealth of sequence information will be productive for conservation studies, selective breeding and designing future strategies for identifying disease associations.