Genome Sequencer FLX Research Articles

Immunoglobulin Variable Region Gene Sequences Reveal N-Glycosylation Motifs As an Early and Stable Event in Follicular Lymphoma Pathology

Introduction: Follicular lymphoma (FL) cells retain expression of a functional B cell receptor (BCR) despite the loss of one Ig allele due to the hallmark t14:18 translocation and ongoing somatic hypermutation (SHM) of the variable genes (V genes) which increases the likelihood of crippling mutations. SHM introduces N-glycosylation (N-gly) motifs within the V genes, a feature exclusively restricted to germinal centre (GC)-derived lymphomas. Oligosaccharides of the high mannose type are added to motifs and interact with calcium-dependent lectins associated with cells of the microenvironment. This activates the BCR signalling pathway and likely contributes to the survival, retention and proliferation of tumor cells in the GC. Determining at what stage of disease evolution N-gly motifs are acquired and their behaviour during progression can ascertain their importance in pathogenesis and their potential as an effective therapeutic target. To achieve this, we analysed motifs within the Ig heavy chain variable gene (IGHV) of tumor-related subclones across temporal FL samples.Method: Genomic DNA from three FL patients taken at different time points of disease progression were analysed. In total, 8 samples were selected, all carrying an IGHV3 rearranged tumor clone. IGHV DNA amplicons were sent for 2x250bp paired-end sequencing using the Miseq Illumina platform (Genewiz, NJ). Tumor-related reads with counts greater than ten were selected following analysis on IMGT/HIGH-V-QUEST. Reads were aligned and unique sequences were assigned as subclones. Additional tumor related reads sequenced on the Roche 454 Life Sciences Genome Sequencer FLX were available (Patients 4 & 5). Subclones were analysed for N-gly motifs and evolutionary pathways were generated using the IgTree program, based on intraclonal SHM profiles and homology of tumor clones to the germline IGHV sequence.Results: The earliest time point samples for Patient's 1, 3, 4 & 5 contained one N-gly site within the IGHV of the MC defined by the largest count number. These sites were conserved in >97% of unique subclones (p<0.0001) despite variations in the nucleotide sequence within the region as a result of ongoing SHM. This conservation included the most mutated subclones which had a mean SHM rate of 16.56%. Conservation was maintained across disease events. Patient 2 contained four N-gly sites located within the CDR1, FR2, CDR2, and FR3 regions. The first three sites were conserved in >97% of subclones in and across disease events, whereas the FR3 site was conserved in 95.5% of the diagnostic subclones and in ~80% of the relapsed and transformed populations. No subclones with loss of all four sites were detected for Patient 2. Patient 5 samples were taken from different anatomical sites with tumor populations acquiring distinct N-gly motifs, suggesting an early divergence in tumor evolution. Despite this, a minor population of motif positive clones are shared, suggesting a trafficking ability of subclones. Subclones with motifs made up ≥99% of the total tumor count, highlighting the motif as a feature of the tumor bulk, with motif negative clones representing a minor population. These negative clones are presumably lost during disease progression as they not shared between events. Evolutionary analysis revealed no additional sites are gained as motif positive clones expand while rare negative subclones cannot reacquire sites and do not undergo further diversification.Conclusion: We report for the first time that acquired N-gly motif sites are a clonal feature in FL disease as seen through their conservation both in the heterogeneous subclonal population and the overall tumor mass. The sites are also retained in progression-associated subclones while rare motif-negative subclones disappear. This suggests that although acquisition of additional driver mutations may dampen the tumor's microenvironment dependency, the motifs and added mannoses may retain functional significance at later stages of disease.The data indicates motifs as being a universal event of the reservoir cell pool responsible for propagating disease episodes. Targeting N-gly sites and their interacting partners may lead to the disruption of an early and vital FL-microenvironment interaction, presumably mediated through the mannose-lectin interaction, reducing relapse rates and progression of disease. [Display omitted] DisclosuresForconi:Abbvie: Consultancy; Janssen-Cilag: Consultancy. Gribben:Abbvie: Honoraria; Roche: Honoraria; Pharmacyclics: Honoraria; Novartis: Honoraria; Cancer Research UK: Research Funding; Wellcome Trust: Research Funding; Acerta Pharma: Honoraria, Research Funding; TG Therapeutics: Honoraria; NIH: Research Funding; Kite: Honoraria; Janssen: Honoraria, Research Funding; Unum: Equity Ownership; Medical Research Council: Research Funding; Celgene: Consultancy, Honoraria, Research Funding.

Characteristics Peripheral Blood IgG and IgM Heavy Chain Complementarity Determining Region 3 Repertoire before and after Immunization with Recombinant HBV Vaccine.

Immunization with recombinant HBV vaccine induces specific immune responses in human causing B lymphocytes to produce protective HBsAb, and to form memory B lymphocytes, thereby facilitating HBV immunity in the body. However, B lymphocytes heterogeneity and characteristics are not fully elucidated. In this study, we conducted high-throughput sequencing of BCR heavy chain CDR3 repertoires in 3 healthy volunteers before and after the third immunization with recombinant HBV vaccine. We used Roche 454 Genome Sequencer FLX system to perform a comparative analysis of IgM and IgG H chain CDR3 repertoires. First, we found that the diversity of IgG H chain CDR3 repertoires was 1/6 of IgM on average. Moreover, after the third immunization with HBV vaccine, the diversity of IgG H chain CDR3 repertoires was 1/26 of IgM on average. Second, we detected relatively high levels of HBsAbs in all the healthy volunteers after immunization with HBV vaccine. The volunteers shared a small number of CDR3 sequences before and after immunization, and among each other. However, we did not find completely identical BCR H chain CDR3 amino acid sequences in these volunteers. Lastly, after immunization with recombinant HBV vaccine, the volunteers showed high frequency of IgG H chain CDR3 amino acid sequences mostly resulting from rearrangements of IGHV, IGHD and IGHJ, suggesting that the mechanism of high frequency CDR3 generation might be associated with the maturation of IgG affinity (somatic hypermutation) during the recombinant HBV vaccine-induced B lymphocyte responses. This study identified the characteristics and changes of BCR CDR3 repertoires before and after immunization with HBV vaccine, and evaluated the performance of the sequencing technology for this application. Our findings provide a basis for further research in B lymphocyte generated HBsAb heterogeneity and monitoring the maintenance of memory B lymphocytes.

Genome sequencing and analysis of a granulovirus isolated from the Asiatic rice leafroller, Cnaphalocrocis medinalis.

The complete genome of Cnaphalocrocis medinalis granulovirus (CnmeGV) from a serious migratory rice pest, Cnaphalocrocis medinalis (Lepidoptera: Pyralidae), was sequenced using the Roche 454 Genome Sequencer FLX system (GS FLX) with shotgun strategy and assembled by Roche GS De Novo assembler software. Its circular double-stranded genome is 111,246 bp in size with a high A+T content of 64.8% and codes for 118 putative open reading frames (ORFs). It contains 37 conserved baculovirus core ORFs, 13 unique ORFs, 26 ORFs that were found in all Lepidoptera baculoviruses and 42 common ORFs. The analysis of nucleotide sequence repeats revealed that the CnmeGV genome differs from the rest of sequenced GVs by a 23 kb and a 17kb gene block inversions, and does not contain any typical homologous region (hr) except for a region of non-hr-like sequence. Chitinase and cathepsin genes, which are reported to have major roles in the liquefaction of the hosts, were not found in the CnmeGV genome, which explains why CnmeGV infected insects do not show the phenotype of typical liquefaction. Phylogenetic analysis, based on the 37 core baculovirus genes, indicates that CnmeGV is closely related to Adoxophyes orana granulovirus. The genome analysis would contribute to the functional research of CnmeGV, and would benefit to the utilization of CnmeGV as pest control reagent for rice production.

Association of obesity with serum leptin, adiponectin, and serotonin and gut microflora in beagle dogs.

BackgroundSerotonin (5‐hydroxytryptamine, 5HT) is involved in hypothalamic regulation of energy consumption. Also, the gut microbiome can influence neuronal signaling to the brain through vagal afferent neurons. Therefore, serotonin concentrations in the central nervous system and the composition of the microbiota can be related to obesity.ObjectiveTo examine adipokine, and, serotonin concentrations, and the gut microbiota in lean dogs and dogs with experimentally induced obesity.AnimalsFourteen healthy Beagle dogs were used in this study.MethodsSeven Beagle dogs in the obese group were fed commercial food ad libitum, over a period of 6 months to increase their weight and seven Beagle dogs in lean group were fed a restricted amount of the same diet to maintain optimal body condition over a period of 6 months. Peripheral leptin, adiponectin, 5HT, and cerebrospinal fluid (CSF‐5HT) levels were measured by ELISA. Fecal samples were collected in lean and obese groups 6 months after obesity was induced. Targeted pyrosequencing of the 16S rRNA gene was performed using a Genome Sequencer FLX plus system.ResultsLeptin concentrations were higher in the obese group (1.98 ± 1.00) compared to those of the lean group (1.12 ± 0.07, P = .025). Adiponectin and 5‐hydroytryptamine of cerebrospinal fluid (CSF‐5HT) concentrations were higher in the lean group (27.1 ± 7.28) than in the obese group (14.4 ± 5.40, P = .018). Analysis of the microbiome revealed that the diversity of the microbial community was lower in the obese group. Microbes from the phylum Firmicutes (85%) were predominant group in the gut microbiota of lean dogs. However, bacteria from the phylum Proteobacteria (76%) were the predominant group in the gut microbiota of dogs in the obese group.Conclusions and Clinical ImportanceDecreased 5HT levels in obese group might increase the risk of obesity because of increased appetite. Microflora enriched with gram‐negative might be related with chronic inflammation status in obese dogs.

Draft Genome Sequences of Streptococcus bovis Strains ATCC 33317 and JB1

We report the draft genome sequences of Streptococcus bovis strain ATCC 33317 (CVM42251) isolated from cow dung and strain JB1 (CVM42252) isolated from a cow rumen in 1977. The strains were sequenced using the Genome Sequencer FLX 454 system. The genome sizes are approximately 2 Mb and 2.2 Mb, respectively.

Successful heterologous expression of a novel chitinase identified by sequence analyses of the metagenome from a chitin-enriched soil sample

Chitin and its derivative chitosan are abundant natural polysaccharides with many potential industrial applications. Metagenomic analysis of chitin-enriched soil samples using the Roche Genome Sequencer FLX platform led to the identification of several novel genes for chitin and chitosan modifying enzymes (CCMEs) which may be used to produce novel chitosans. The sequencing approach yielded 2,281,090 reads with an average length of 378bp amounting to a total sequence information of approximately 851Mb. Assembly of the obtained sequences comprised 699,710 reads representing 30.68% of all reads. A total of 6625 contigs larger than 500bp containing 16,289 predicted genes are included in the assembly. Taxonomic profiling of the indigenous microbial community by applying the software CARMA revealed that 96.1% of the reads were of bacterial origin including 17% assigned to the family Xanthomonadaceae. Several putative genes encoding CCMEs were identified by comparison against the GenBank database, inclusive a full-length chitinase gene which was codon optimized for Escherichia coli and heterologously synthesized as a Strep-tagged protein in E. coli Rosetta 2 using the pET vector system. Approximately 5mg of the novel active chitinase was purified as demonstrated by dot assay analysis using glycol chitin as a substrate. Next generation metagenomic sequencing, thus, emerges as a new and powerful tool for the identification of potentially novel biocatalysts of biotechnological value.

Single origin of three plasmids bearing blaCTX-M-15 from different Klebsiella pneumoniae clones.

To determine and compare the complete nucleotide sequences of plasmids carrying blaCTX-M-15 from three different Klebsiella pneumoniae clones. IncFII-type plasmids pKP02022, pKP09085 and pKP007 were extracted from three K. pneumoniae strains. These strains belong to sequence types (STs) ST15, ST48 and ST23, respectively, and were isolated in Korea. Plasmids were sequenced using the 454 Genome Sequencer FLX system. The three plasmids, pKP02022 (203577 bp), pKP09085 (213019 bp) and pKP007 (246 176 bp), all exhibited a very similar structure, with a pKPN3-like backbone and a resistance region including blaOXA-1, aac(6')-Ib-cr and cat genes as well as blaCTX-M-15. They were also very similar to pUUH239.2, previously isolated in Sweden. Iron (III) uptake-related genes were found in pKP007 from the ST23 strain, which has been reported to be associated with liver abscesses. The resistance region contained several insertion sequences, such as IS26, which may play an important role in structural rearrangements of plasmids. The very similar structure of the three plasmids, extracted from different clones, suggests that the spread of CTX-M-producing K. pneumoniae isolates might result from the horizontal transfer of plasmids and subsequent integration and recombination.

Taxonomic Profiling and Metagenome Analysis of a Microbial Community from a Habitat Contaminated with Industrial Discharges.

Industrial units, manufacturing dyes, chemicals, solvents, and xenobiotic compounds, produce liquid and solid wastes, which upon conventional treatment are released in the nearby environment and thus are the major cause of pollution. Soil collected from contaminated Kharicut Canal bank (N 22°57.878'; E 072°38.478'), Ahmedabad, Gujarat, India was used for metagenomic DNA preparation to study the capabilities of intrinsic microbial community in dealing with xenobiotics. Sequencing of metagenomic DNA on the Genome Sequencer FLX System using titanium chemistry resulted in 409,782 reads accounting for 133,529,997 bases of sequence information. Taxonomic analyses and gene annotations were carried out using the bioinformatics platform Sequence Analysis and Management System for Metagenomic Datasets. Taxonomic profiling was carried out by three different complementary approaches: (a) 16S rDNA, (b) environmental gene tags, and (c) lowest common ancestor. The most abundant phylum and genus were found to be "Proteobacteria" and "Pseudomonas," respectively. Metagenome reads were mapped on sequenced microbial genomes and the highest numbers of reads were allocated to Pseudomonas stutzeri A1501. Assignment of obtained metagenome reads to Gene Ontology terms, Clusters of Orthologous Groups of protein categories, protein family numbers, and Kyoto Encyclopedia of Genes and Genomes hits revealed genomic potential of indigenous microbial community. In total, 157,024 reads corresponded to 37,028 different KEGG hits, and amongst them, 11,574 reads corresponded to 131 different enzymes potentially involved in xenobiotic biodegradation. These enzymes were mapped on biodegradation pathways of xenobiotics to elucidate their roles in possible catalytic reactions. Consequently, information obtained from the present study will act as a baseline which, subsequently along with other "-omic" studies, will help in designing future bioremediation strategies in effluent treatment plants and environmental clean-up projects.

Abstract 1505: Towards an understanding of the different expression profile of fibroblasts from distinct subtypes from breast cancer and normal mammary tissue assessed by transcriptome sequencing.

Abstract The highly heterogeneous clinical, histological, biological and genetic nature of breast malignancies is due in part to their extreme molecular complexity. During breast carcinogenesis, cancer associated fibroblasts (CAFs) have active role in the initiation, progression, metastasis and recurrence of tumors, which adds new levels of complexity to cancer biology, but also brings new prognostic and therapeutic opportunities. We implemented a pilot study to explore the postulation that CAFs within the stroma may exhibit specific gene expression profiles according to the carcinoma subtype. We enrolled frozen breast tissues from nine human breast samples representing three normal tissues, three luminal A and three triple negative subtypes. Fibroblasts were isolated by laser microdissection. For each subtype studied, a cDNA library was constructed and submitted to a transcriptome sequencing (RNA-Seq) on the Genome Sequencer FLX Titanium 454 Plataform. The generated data revealed a total of 14,950 transcripts sequenced and 10,586 genes identified (53.72% of the human genome repertoire). A list of 221 differentially expressed transcripts, obtained by pair wise comparisons among the 3 groups, was ranked by their probabilities of differential expression from 91%-100% (greater expression probability), and from 0.09-9.8% (lower expression probability). Functional similarity interactions (Fun-Net) were constructed using Gene Ontology (GO) annotations as defined in the Entrez Gene database to identify biological functions overrepresented. In addition, 36 differentially expressed genes were selected for technical and biological validation by RT-qPCR (Taqman Low Density Array - TLDA) and tissue microarray (TMA) techniques. Until now 10 out of 20 differentially expressed genes were validated by RT-qPCR. Considering the comparison between fibroblasts associated either to triple negative or luminal breast carcinoma subtypes, five differentially expressed genes were confirmed: S100A13, PPIA, HNRNP0, CUL3 and SLC25A32. Our results indicate that specific changes exist in CAFs derived from breast cancer subtypes, which in turn may contribute to each tumor subtype specific clinical behavior. Supported by FAPESP: 05/60333-7, 09/10088-7. Citation Format: Natália Bromberg, Dirce Maria Carraro, Carlos Alberto Bragança Pereira, Gustavo Campos Molina, Mabel Gigliola Pinilla, Elisa Napolitano Ferreira, Maria Mitzi Brentani. Towards an understanding of the different expression profile of fibroblasts from distinct subtypes from breast cancer and normal mammary tissue assessed by transcriptome sequencing. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1505. doi:10.1158/1538-7445.AM2013-1505

Murine Gut Microbiota and Transcriptome Are Diet Dependent

Here, we determine how formula feeding impacts the gut microbiota and host transcriptome. Formula-fed (FF) infants are at risk for diseases that involve complex interactions between microbes and host immune elements such as necrotizing enterocolitis. The aims of this study were to simultaneously examine the microbiota and host transcriptional profiles of FF and maternal-fed (MF) mice to evaluate how diet impacts gut colonization and host genes. After 72 hours of FF or MF, colonic tissue was collected. 16S ribosomal RNA was sequenced with Roche GS-FLX (Genome Sequencer-FLX) pyrosequencing. Operational taxonomical unit clustering, diversity analysis, and principal coordinate analysis (PCA) were performed. Complementary DNA libraries were sequenced by Solexa. Reads were annotated by BLAST (Basic Local Alignment Search Tool) search against mouse RNA database [National Center for Biotechnology Information (NCBI) build-37] and functionally classified using the KOG (Eukaryotic Orthologous Groups) database (NCBI). Firmicutes (P < 0.001) was the dominant phylum in MF pups, whereas Proteobacteria (P < 0.001) and Bacteroidetes (P < 0.05) were dominant in FF mice. On the genus level, FF mice had increased Serratia (P < 0.001) and Lactococcus (P < 0.05) whereas MF mice had increased Lactobacillus (P < 0.001). PCA confirmed clustering by diet. Solexa sequencing demonstrated different (P < 0.05) messenger RNA transcript levels in 148 genes. Heme oxygenase 1 (P < 0.01), an oxidative stress marker, was increased 25-fold in FF mice. In addition, decreased vinculin (P < 0.05), a cytoskeletal protein associated with adherens junctions in FF pups suggested impaired gut structural integrity. Diet also impacted immune regulation, cell cycle control/gene expression, cell motility, and vascular function genes. FF shifted gut microbiota and structural integrity, oxidative stress, and immune function genes, presumably increasing vulnerability to disease in FF mice. Interrogation of microbial and host gene expression in FF neonates may offer new insight on how diet affects disease pathogenesis.

Allele Identification for Transcriptome-Based Population Genomics in the Invasive PlantCentaurea solstitialis

Transcriptome sequences are becoming more broadly available for multiple individuals of the same species, providing opportunities to derive population genomic information from these datasets. Using the 454 Life Science Genome Sequencer FLX and FLX-Titanium next-generation platforms, we generated 11−430 Mbp of sequence for normalized cDNA for 40 wild genotypes of the invasive plant Centaurea solstitialis, yellow starthistle, from across its worldwide distribution. We examined the impact of sequencing effort on transcriptome recovery and overlap among individuals. To do this, we developed two novel publicly available software pipelines: SnoWhite for read cleaning before assembly, and AllelePipe for clustering of loci and allele identification in assembled datasets with or without a reference genome. AllelePipe is designed specifically for cases in which read depth information is not appropriate or available to assist with disentangling closely related paralogs from allelic variation, as in transcriptome or previously assembled libraries. We find that modest applications of sequencing effort recover most of the novel sequences present in the transcriptome of this species, including single-copy loci and a representative distribution of functional groups. In contrast, the coverage of variable sites, observation of heterozygosity, and overlap among different libraries are all highly dependent on sequencing effort. Nevertheless, the information gained from overlapping regions was informative regarding coarse population structure and variation across our small number of population samples, providing the first genetic evidence in support of hypothesized invasion scenarios.

MiRNA Expression Profile Analysis in Kidney of Different Porcine Breeds

microRNAs (miRNAs) are important post-transcriptional regulators in eukaryotes that target mRNAs repressing their expression. The uncertain process of pig domestication, with different origin focuses, and the selection process that commercial breeds suffered, have generated a wide spectrum of breeds with clear genetic and phenotypic variability. The aim of this work was to define the miRNAs expression profile in kidney of several porcine breeds. Small RNA libraries from kidney were elaborated and high-throughput sequenced with the 454 Genome Sequencer FLX (Roche). Pigs used were classified into three groups: the European origin group (Iberian breed and European Wild Boar ancestor), European commercial breeds (Landrace, Large White and Piétrain breeds) and breeds with Asian origin (Meishan and Vietnamese breeds). A total of 229 miRNAs were described in the pig kidney miRNA profile, including 110 miRNAs out of the 257 previously described pig miRNAs and 119 orthologous miRNAs. The most expressed miRNAs in pig kidney microRNAome were Hsa-miR-200b-3p, Ssc-miR-125b and Ssc-miR-23b. Moreover, 5 novel porcine miRNAs and 3 orthologous miRNAs could be validated through RT-qPCR. miRNA sequence variation was determined in 116 miRNAs, evidencing the presence of isomiRs. 125 miRNAs were differentially expressed between breed groups. The identification of breed-specific miRNAs, which could be potentially associated to certain phenotypes, is becoming a new tool for the study of the genetic variability underlying complex traits and furthermore, it adds a new layer of complexity to the interesting process of pig evolution.

Establishment and interpretation of the genome sequence of the phytopathogenic fungus Rhizoctonia solani AG1-IB isolate 7/3/14

Anastomosis group AG1-IB isolates of the anamorphic basidiomycetous fungus Rhizoctonia solani Kühn affect various agricultural and horticultural important crops including bean, rice, soybean, figs, hortensia, cabbage and lettuce. To gain insights into the genome structure and content, the first draft genome sequence of R. solani AG1-IB isolate 7/3/14 was established. Four complete runs on the Genome Sequencer (GS) FLX platform (Roche Applied Science) yielding approx. a 25-fold coverage of the R. solani genome were accomplished. Assembly of the sequence reads by means of the gsAssembler software version 2.6 applying the heterozygotic mode resulted in numerous contigs and scaffolds and a predicted size of 87.1Mb for the diploid status of the genome. ‘Contig-length vs. read-count’ analysis revealed that the assembled contigs can be classified into five different groups. Detailed BLAST-analysis revealed that most contigs of group II feature high-scoring matches to other contigs of the same group suggesting that distinguishable allelic variants exist for many genes. Due to the supposed diploid and heterokaryotic nature of R. solani AG1-IB 7/3/14, this result has been anticipated. However, the heterokaryotic character of the isolate is not really supported by sequencing data obtained for the isolate R. solani AG1-IB 7/3/14. Coverage of group III contigs is twice as high as for group II contigs which can also be explained by the diploid status of the genome and indistinguishable alleles on homologous chromosomes. Assembly of sequence data led to the identification of the rRNA unit (group V contigs) and the mitochondrial (mt) genome (group IV contigs) which is a circular molecule of 162,751bp in size featuring a GC-content of 36.4%. The R. solani 7/3/14 mt-genome is one of the largest fungal mitochondrial genomes known to date. Its large size essentially is due to the presence of numerous non-conserved hypothetical ORFs and introns. Gene prediction for the R. solani AG1-IB 7/3/14 genome was conducted by the Augustus Gene Prediction Software for Eukaryotes (version 2.6.) applying the parameter set for the fungus Coprinopsis cinerea okayama 7#130. Gene prediction and annotation provided first insights into the R. solani AG1-IB 7/3/14 gene structure and content. In total, 12,422 genes were predicted. The average number of exons per gene is five. Exons have a mean length of 214bp, whereas introns on average are 66bp in length. Annotation of the genome revealed that 4169 of 12,422 genes could be assigned to KOG functional categories.

Sequence of a fusogenic herpes simplex virus, RH2, for oncolytic virotherapy

RH2 is a novel oncolytic herpes simplex virus type 1 (HSV-1) produced by simultaneous infection with neurovirulent γ134.5 gene-deficient HSV-1 R849 derived from strain F and the spontaneously occurring, fusogenic HSV-1 HF in cell culture. The genome of RH2 was studied using Genome Sequencer FLX. RH2 comprised 149 64 bp and it was shown that the lacZ gene was inserted into the γ134.5 gene of R849. Comparison of ORFs revealed that RH2 had 100 % identity with strain F in 21/58 unique long (UL) genes (36.2%) and 1/13 unique short (US) genes (7.7%). RH2 had 100% amino acid identity with HF10 in 24/58 UL genes (41.4%) and 9/13 US genes (69.2%). Twelve genes, including UL27 (gB), US4 (gG) and UL6 (gD), had amino acid changes unique to RH2. Amino acid changes in gB occurred at positions 459 (T→A) and 817 (L→P). Other unique features were the amino acids missing in UL36 (VP1/2) and UL46 (VP11/12). Thus, RH2 is an HF10-based vector preserving the fusogenic amino acid changes of gB but lacking the γ134.5 gene. RH2 is expected to be a version of HF10 useful for the treatment of brain tumours as well as oral squamous cell carcinoma. Spontaneously occurring HSV-1 mutants may also be useful clinically, as their genome sequences can easily be determined by this genome sequencing system.

A high-throughput next-generation sequencing-based method for detecting the mutational fingerprint of carcinogens

Many carcinogens leave a unique mutational fingerprint in the human genome. These mutational fingerprints manifest as specific types of mutations often clustering at certain genomic loci in tumor genomes from carcinogen-exposed individuals. To develop a high-throughput method for detecting the mutational fingerprint of carcinogens, we have devised a cost-, time- and labor-effective strategy, in which the widely used transgenic Big Blue® mouse mutation detection assay is made compatible with the Roche/454 Genome Sequencer FLX Titanium next-generation sequencing technology. As proof of principle, we have used this novel method to establish the mutational fingerprints of three prominent carcinogens with varying mutagenic potencies, including sunlight ultraviolet radiation, 4-aminobiphenyl and secondhand smoke that are known to be strong, moderate and weak mutagens, respectively. For verification purposes, we have compared the mutational fingerprints of these carcinogens obtained by our newly developed method with those obtained by parallel analyses using the conventional low-throughput approach, that is, standard mutation detection assay followed by direct DNA sequencing using a capillary DNA sequencer. We demonstrate that this high-throughput next-generation sequencing-based method is highly specific and sensitive to detect the mutational fingerprints of the tested carcinogens. The method is reproducible, and its accuracy is comparable with that of the currently available low-throughput method. In conclusion, this novel method has the potential to move the field of carcinogenesis forward by allowing high-throughput analysis of mutations induced by endogenous and/or exogenous genotoxic agents.

Abstract 4271: Fibroblast transcriptome sequencing reveals different gene expression profiles among distinct subtypes of breast cancer and normal mammary tissue

Abstract Cancer associated fibroblasts, CAFs, is believed to actively participate in the growth and invasion of tumor cells by providing a unique tumor microenvironment, rendering increasingly clear that the development of cancer cannot be dissociated from its local microenvironment. However, neither the origin of CAFs nor the criteria to distinguish CAFs from normal fibroblasts has been well established. Moreover, the complex nature of the heterogeneity that exists both within and between breast cancers, particularly as related to gene expression profiling, needs more recognition. It must be appreciated that CAFs from different subtypes of breast cancers have distinct gene expression profiles as well as do CAFs in comparison to normal breast fibroblasts (NAFs). The identification of these differentially expressed genes (DEG) may shed light on the molecular interactions of fibroblasts with carcinoma cells and to further understand their signaling pathways that are actively involved. The advent of next generation sequence technologies and the realization that breast cancer is heterogeneous has shifted the focus to the identification of molecular markers of prognostic relevance. In the present study, we isolated total RNA from fibroblasts of frozen tissue samples of human invasive ductal breast carcinoma luminal A and triple negative subtypes and also from normal surrounding tissue from fibroadenoma lesions using laser capture microdissection. Preparing 3′ fragments cDNA libraries from fibroblast samples and sequencing on the Genome Sequencer FLX Titanium 454 Plataform, we obtained a transcriptional snapshot of CAFs and NAF. Sequencing and filtering (removal of adapter, polyA tail etc) of these sequences resulted in a total of 376,528 high quality reads with an average length of 159.43 bp. A total of 14,950 transcripts were sequenced and corresponded to 10,586 genes, representing 53.72% of the human genome repertoire. The analysis of DEG between the distinct samples was investigated by test on proportions (p value ≤ 0.05 and 2.0 fold change difference in expression level). The comparison of expression in NAFs versus triple negative CAF yielded 192 DEG while NAF versus luminal A CAF revealed 88 DEG and triple negative CAF versus luminal A CAF presented 41 DEG. Gene ontology (GO) assignments and KEGG pathways classifications were performed to gain an overview of the biological processes in which these DEG are involved. The results suggest that highly specific genes are expressed in each breast cancer fibroblast subtype. Taken all together, genes will be selected for validation by RT-qPCR and tissue microarray using independent samples. Further understanding of detailed functional mechanisms of the differentially expressed genes, may thus yield additional candidates for prognostic and therapeutic intervention. Supported by FAPESP:05/60333-7, 09/10088-7. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4271. doi:1538-7445.AM2012-4271

Insights into the completely annotated genome of Lactobacillus buchneri CD034, a strain isolated from stable grass silage

Lactobacillus buchneri belongs to the group of heterofermentative lactic acid bacteria and is a common member of the silage microbiome. Here we report the completely annotated genomic sequence of L. buchneri CD034, a strain isolated from stable grass silage. The whole genome of L. buchneri CD034 was sequenced on the Roche Genome Sequencer FLX platform. It was found to consist of four replicons, a circular chromosome, and three plasmids. The circular chromosome was predicted to encode 2319 proteins and contains a genomic island and two prophages which significantly differ in G+C-content from the remaining chromosome. It possesses all genes for enzymes of a complete phosphoketolase pathway, whereas two enzymes necessary for glycolysis are lacking. This confirms the classification of L. buchneri CD034 as an obligate heterofermentative lactic acid bacterium. A set of genes considered to be involved in the lactate degradation pathway and genes putatively involved in the breakdown of plant cell wall polymers were identified. Moreover, several genes encoding putative S-layer proteins and two CRISPR systems, belonging to the subclasses I-E and II-A, are located on the chromosome. The largest plasmid pCD034-3 was predicted to encode 57 genes, including a putative polysaccharide synthesis gene cluster, whereas the functions of the two smaller plasmids, pCD034-1 and pCD034-2, remain cryptic. Phylogenetic analysis based on sequence comparison of the conserved marker gene rpoA reveals that L. buchneri CD034 is more closely related to Lactobacillus hilgardii strains than to Lactobacillus brevis and Lactobacillus plantarum strains. Comparison of the L. buchneri CD034 core genome to other fully sequenced and closely related members of the genus Lactobacillus disclosed a high degree of conservation between L. buchneri CD034 and the recently sequenced L. buchneri strain NRRL B-30929 and a more distant relationship to L. buchneri ATCC 11577 and L. brevis ssp. gravesensis ATCC 27305, which cluster together with L. hilgardii type strain ATCC 8290. L. buchneri CD034 genome information will certainly provide the basis for further postgenome studies with the objective to optimize application of the strain in silage production.

Detection of diverse aquatic microbes in blood and organs of drowning victims: First metagenomic approach using high-throughput 454-pyrosequencing

Current 454-pyrosequencing technology enables massive parallel sequencing. We used this technology to investigate the diversity of aquatic microbes in 14 specimens (blood and organs) of two drowning victims and in two water samples taken from the discovery sites. The 16S ribosomal RNA (rRNA) genes of microbes, which are often used to identify species (or genera), have nine highly variable regions (V1-V9), each of which is surrounded by conserved regions. Some parts within the conserved regions are common over domains of microbes, such as between bacteria and algae (16S rRNA genes on algal chloroplast genomes). We therefore simultaneously amplified the target regions (V7 and V8) of various microbes in the blood and organs of drowning victims using PCR with custom-designed primers that were based on the conserved regions. We then exhaustively analyzed the PCR products by pyrosequencing using the Genome Sequencer FLX Titanium system (Roche-454 Life Sciences). This approach identified a wide array of bacteria including cyanobacteria and algae including Bacillariophyceae (diatom), Cryptophyceae, Dictyochophyceae, Chrysophyceae and Trebouxiophyceae in the blood and organs of the victims and water at discovery sites. Our data further indicated that when conventional diatom testing of lungs yielded insufficient evidence of water aspiration, the detection of various exogenous microbes by 454-pyrosequencing is very useful to support a conclusion of death by drowning. To the best of our knowledge, this is the first attempt to use a new generation sequencer to investigate diverse aquatic microbes in the blood and closed organs of drowning victims.

Profiling of the metabolically active community from a production-scale biogas plant by means of high-throughput metatranscriptome sequencing

Structural composition and gene content of a biogas-producing microbial community from a production-scale biogas plant fed with renewable primary products was recently analyzed by means of a metagenome sequencing approach. To determine the transcriptionally active part of the same biogas community and to identify key transcripts for the biogas production process, the metatranscriptome of the microorganisms was sequenced for the first time. The metatranscriptome sequence dataset generated on the Genome Sequencer FLX platform is represented by 484,920 sequence reads. Taxonomic profiling of the active part of the community by classification of 16S ribosomal sequence tags revealed that members of the Euryarchaeota and Firmicutes account for the dominant phyla. Only smaller fractions of the 16S ribosomal sequence tags were assigned to the phyla Bacteroidetes, Actinobacteria and Synergistetes. Among the mRNA-derived sequence tags from the metatranscriptome dataset, transcripts encoding enzymes involved in substrate hydrolysis, acidogenesis, acetate formation and methanogenesis could be identified. Transcripts for enzymes functioning in methanogenesis are among the most abundant mRNA tags indicating that the corresponding pathway is very active in the methanogenic sub-community. As a frame of reference for evaluation of metatranscriptome sequence data, the 16S rDNA-based taxonomic profile of the community was analyzed by means of high-throughput 16S rDNA amplicon sequencing. Processing of the obtained amplicon reads resulted in 18,598 high-quality 16S rDNA sequences covering the V3-V4 hypervariable region of the 16S rRNA gene. Comparison of the taxonomic profiles deduced from 16S rDNA amplicon sequences and the metatranscriptome dataset indicates a high transcriptional activity of archaeal species. Overall, it was shown that the most abundant species dominating the community also contributed the majority of the transcripts. In the future, key transcripts for the biogas production process will provide valuable markers for evaluation of the performance of biogas-producing microbial communities with the objective to optimize the biotechnology of this process.

Quasispecies dynamics in main core epitopes of hepatitis B virus by ultra-deep-pyrosequencing

To investigate the variability of the main immunodominant motifs of hepatitis B virus (HBV) core gene by ultra-deep-pyrosequencing (UDPS). Four samples (2 genotype A and 2 genotype D) from 4 treatment-naïve patients were assessed for baseline variability. Two additional samples from one patient (patient 4, genotype D) were selected for analysis: one sample corresponded to a 36-mo treatment-free period from baseline and the other to the time of viral breakthrough after 18 mo of lamivudine treatment. The HBV region analyzed covered amino acids 40 to 95 of the core gene, and included the two main epitopic regions, Th50-69 and B74-84. UDPS was carried out in the Genome Sequencer FLX system (454 Life Sciences, Roche). After computer filtering of UDPS data based on a Poisson statistical model, 122,813 sequences were analyzed. The most conserved position detected by UDPS was analyzed by site-directed mutagenesis and evaluated in cell culture. Positions with highest variability rates were mainly located in the main core epitopes, confirming their role as immune-stimulating regions. In addition, the distribution of variability showed a relationship with HBV genotype. Patient 1 (genotype A) presented the lowest variability rates and patient 2 (genotype A) had 3 codons with variability higher than 1%. Patient 3 and 4 (both genotype D) presented 5 and 8 codons with variability higher than 1%, respectively. The median baseline frequencies showed that genotype A samples had higher variability in epitopic positions than in the other positions analyzed, approaching significance (P = 0.07, sample 1 and P = 0.05, sample 2). In contrast, there were no significant differences in variability between the epitopic and other positions in genotype D cases. Interestingly, patient 1 presented a completely mutated motif from amino acid 64 to 67 (E₆₄LMT₆₇), which is commonly recognized by T helper cells. Additionally, the variability observed in all 4 patients was particularly associated with the E₆₄LMT₆₇ motif. Codons 78 and 79 were highly conserved in all samples, in keeping with their involvement in the interaction between the HBV virion capsid and the surface antigens (HBsAg). Of note, codon 76 was even more conserved than codons 78 and 79, suggesting a possible role in HBsAg interactions or even in hepatitis B e antigen conformation. Sequential analysis of samples from patient 4 (genotype D) illustrated the dynamism of the HBV quasispecies, with strong selection of one minor baseline variant coinciding with a decrease in core variability during the treatment-free and lamivudine-treated period. The drop in variability seemed to result from a "steady state" situation of the HBV quasispecies after selection of the variant with greatest fitness. Host immune pressure seems to be the main cause of HBV core evolution. UDPS analysis is a useful technique for studying viral quasispecies.