Roche GS FLX Research Articles

De novo analysis and functional classification of the transcriptome of the root lesion nematode, Pratylenchus thornei, after 454 GS FLX sequencing

The migratory endoparasitic root lesion nematode Pratylenchus thornei is a major pest of the cereals wheat and barley. In what we believe to be the first global transcriptome analysis for P. thornei, using Roche GS FLX sequencing, 787,275 reads were assembled into 34,312 contigs using two assembly programs, to yield 6,989 contigs common to both. These contigs were annotated, resulting in functional assignments for 3,048. Specific transcripts studied in more detail included carbohydrate active enzymes potentially involved in cell wall degradation, neuropeptides, putative plant nematode parasitism genes, and transcripts that could be secreted by the nematode. Transcripts for cell wall degrading enzymes were similar to bacterial genes, suggesting that they were acquired by horizontal gene transfer. Contigs matching 14 parasitism genes found in sedentary endoparasitic nematodes were identified. These genes are thought to function in suppression of host defenses and in feeding site development, but their function in P. thornei may differ. Comparison of the common contigs from P. thornei with other nematodes showed that 2,039 were common to sequences of the Heteroderidae, 1,947 to the Meloidogynidae, 1,218 to Radopholus similis, 1,209 matched expressed sequence tags (ESTs) of Pratylenchus penetrans and Pratylenchus vulnus, and 2,940 to contigs of Pratylenchus coffeae. There were 2,014 contigs common to Caenarhabditis elegans, with 15.9% being common to all three groups. Twelve percent of contigs with matches to the Heteroderidae and the Meloidogynidae had no homology to any C. elegans protein. Fifty-seven percent of the contigs did not match known sequences and some could be unique to P. thornei. These data provide substantial new information on the transcriptome of P. thornei, those genes common to migratory and sedentary endoparasitic nematodes, and provide additional understanding of genes required for different forms of parasitism. The data can also be used to identify potential genes to study host interactions and for crop protection.

Functional Characterization of CD4+ T-Cells in Aplastic Anemia (AA)

Abstract 1340We have examined the role of CD4+ T-cells in the pathogenesis of AA in 63 patients, 48 of whom were analyzed at diagnosis and 15 following immunosuppressive therapy (IST). Absolute numbers of CD4+ regulatory T cells (Tregs, defined as CD3+CD4+CD25highCD27+Foxp3+) were lower in pre-treatment AA patients compared to 10 healthy donors (HDs) (5.5 × 106 v 1.4 × 107)(p=0.01). In patients with severe (SAA) and very severe AA (VSAA), the absolute number and frequency of Tregs were lower than non-severe AA (NSAA) (4.4 × 106/L v 1 × 107/L)(p=0.01) and HDs (4.4 × 106/L v 3 × 107/L) (p<0.001). Absolute numbers of Th1 and Th2 cells in all pre-treatment patients were higher compared to HDs (6.4 × 107/L v 1.8 × 107/L)(p=0.03) for Th1 and (2.6 × 107/L v 2.4 × 106/L)(p=0.006) Th2 cells. Although mean percentages of AA Th17 cells were higher than in HDs (1.5% v 0.15%)(p=0.04), differences in absolute numbers were not significant. Absolute numbers of Th2 and Th17 cells were increased in SAA (1.3 × 107/L v 7.4 × 106/L for Th2)(p=0.01) compared to NSAA (5.7 × 106/L v 2.15 × 106/L for Th17)(p=0.02). Ratios of Th1/Tregs (p=0.003), Th2/Tregs (p=0.02), and Th17/Tregs (p=0.001) were higher in SAA and VSAA compared to NSAA. Percentage of both activated (CD4+CD45RA−CD25highFoxp3high) and resting (CD4+CD45RA+ CD25highFoxp3low) Tregs was decreased in AA patients, compared to HDs (p=0.004 and p=0.01), whereas cytokine secreting Tregs (CD4+CD45RA−CD25high Foxp3low) were increased in AA (p<0.003). Sorted Tregs from AA patients did not suppress cytokine secretion by autologous or HD T effectors (Te) cells in 1:1 co-cultures, whereas IL-2 and IFN-γ secretion by AA Te (CD4+CD25lowCD127high) was suppressible by allogeneic Tregs from HDs, confirming Tregs dysfunction. AA Tregs did not inhibit either CD154 or CD69 expression on Te cells. Tregs from AA patients secreted significantly more IFN-γ, TNF-α and IL-17 (p=0.02, p=0.02 and p=0.01, respectively) after 4 hours stimulation with PMA/Ionomycine compared to HDs. Expression levels of FoxP3, ROR□c and T-bet in AA Tregs was normal. IFN-γ secreting cells (Th1) were enriched using enrichment kit then further enriched by FACS sorting. CDR3 region products of TCR Vβ-chain were amplified using Vβ specific forward and Cβ reverse primers. CDR3 PCR products from AA patients and HDs were subjected 454 sequencing (Roche GS FLX titanium). Sequencing was performed to yield an average ‘depth' in excess of 1000 clonally reads (1000x) for each sample specific CDR3 PCR amp icon. Reads were processed using Roche Amp icon Variant Analyzer software (AVA). Diversity of TCR receptors (measured by spectratyping and confirmed by high throughput deep sequencing) in AA Th1 cells was lower than HDs (p=0.037), as shown by the percentage and number of consensus clusters in total sequence reads. Interestingly, percentages of the most dominant CDR3 clones, revealed by high throughput sequencing, were higher in AA compared to HDs, regardless of spectratyping pattern. Global gene expression of Tregs was compared in 3 pre-IST AA patients and 5 HDs. A unique gene signature consisting of 86 genes that were significant was identified. There were 8 down regulated genes (fold change) in the pre-treatment group; PIN4 (−4.1), OR2T12 (−3.3), AMAC1 (−2.73), PERP (−2.69), UTS2 (−2.27), RNF139 (−2.13), COMMD9 (−2.09) and LOC100128356 (−2.01). The top 10 of 78 up-regulated genes in the pre-treatment group were HBB (19.5), PSME2 (13.8), CSDA (13.07), FAM127A (7.78), EXOSC1 (7.73), BPGM (7.43), CYSLTR1 (7.17), CHPT1 (6.96) and PLAC8 (6.71). qPCR analysis for CSDA, HBB, PSMiE2, PERP, PIN4, and UTS2 confirmed a similar trend to the microarray results. Interestingly absolute number of Tregs, and Th2/Treg ratio were higher in 10 IST responsive patients compared to 5 non-responsive patients (p=0.005 and 0.02, respectively).We show that expansion of Th1, Th2, Th17, and decreased/skewed Tregs immunophenotype and function are a consistent and defining feature of SAA and VSAA. Clonal expansion of Th1 cells is likely to be antigen driven and the presence of dysfunctional Tregs aggravates this autoimmune response.Increases of Tregs, and Th2/Treg ratios following IST predicts a favourable response to this treatment. Disclosures:No relevant conflicts of interest to declare.

Kings Health Partners 17 Gene Amplicon Panel for Next Generation Sequencing One Stop Mutational Assessment In Myeloid Malignancies

Abstract 748NCL, AA, AK & AS contributed equally to the studyA large number of acquired mutations have been identified in haematological malignancies in recent years. An increasing number of targets present a new problem for diagnostic molecular pathology laboratories. It is not practical for a laboratory to design increasing numbers of gene specific assays for these targets. For these reasons a single assay capable of detecting a large number of gene mutations is desirable. Here we describe a next generation sequencing approach using the Roche 454 platform which is capable of the simultaneous mutation analysis of 17 genes at an appropriate sensitivity level in multiple patient samples. This methodology allows mutation scanning of all coding exons of 5 complete genes (TP53, EZH2, DNMT3a, RUNX1 and CEBPa) scanning of the mutational hotspots of 5 genes (ASXL1 exon 12, JAK2 exon 12, FLT3, MPL and CBL) and the analysis of specific mutation hotspots in a further 8 genes (NRAS, KRAS, NPM1, IDH1, IDH2, KIT, BRAF, JAK2 exon 14). In order to cover all these regions, a total of 99 PCR amplicons (average length 300bp range 250–400bp) were amplified for each patient sample. To avoid time consuming quantification and normalisation of individual amplicons downstream of PCR, we developed a simple method to first designate the amplicons into 4 groups based on the efficiency of PCR amplification. Following amplification the 1st round PCR products are diluted according to the grouping assigned to the particular amplicon prior to a second round of PCR in which MIDs or “barcodes” are added in a patient specific manner. The products of the 2nd round PCRs are then pooled for each patient and normalised before pooling the entire library for subsequent sequencing. The sequencing is carried out using the Roche GS FLX titanium reagents.To validate this approach we have prepared libraries from 80 AML patients with normal cytogenetics. Sequencing of these patients was split over 3 runs of the FLX instrument using standard conditions recommended by the manufacturer. The average number of reads per amplicon was 300 with 95% of amplicons having a minimum coverage of 200 reads. The approach allows the detection of mutations at a sensitivity of approximately 5%. Mutations were detected in 14 /17 genes in at least one patient sample. No mutations of BRAF, JAK2 or MPL were detected. In line with previously published data, mutations were found frequently in NPM1 (67%), DNMT3a (55%) FLT3 ITD (44%) and less frequently in the remaining genes. These data correlate well with published data for normal karyotype AML. In 6/17 genes the mutation status of the patient samples had been previously analysed using: Fragment analysis (NPM1 and FLT3 ITD), RFLP (FLT3 TKD), Sanger Sequencing (IDH1, IDH2, ASXL1, TP53). Significantly, 12 additional mutations were detected (NPM1 n=2, FLT3 ITD n=2, FLT3 TKD n=4, IDH1 n=2, IDH2 n=2, ASXL1 n=4) using the new methodology demonstrating an improvement in sensitivity over and above individual gene specific assays. A single run of the FLX instrument enables the analysis of 30 patients in a batch. In our hands library preparation, sequencing and analysis of 30 patients using the workflow described takes a skilled operator 10 full days. With an estimated test price of \\P650 ($1000), this compares extremely favourably with the price of providing these analyses separately estimated to be in excess of \\P4000 ($6500).To conclude we have developed a next generation amplicon sequencing approach to assay 17 individual target genes including whole gene coding sequences and more focused mutational hotspots. In addition we have validated a strategy which dramatically reduces the amount of operator time required compared with most amplicon sequencing approaches. This approach offers a highly flexible platform for analysing multiple gene targets in multiple samples. Changes to the targets analysed or the effective sensitivity can easily be incorporated without the need to make major modifications to the procedure. Disclosures:No relevant conflicts of interest to declare.

Analysis of adaptive responses of Pinus pinaster to changing environmental conditions in the Mediterranean region

Analysis of adaptive responses of Pinus pinaster to changing environmental conditions in the Mediterranean region

Advancing Eucalyptus genomics: identification and sequencing of lignin biosynthesis genes from deep-coverage BAC libraries

BackgroundEucalyptus species are among the most planted hardwoods in the world because of their rapid growth, adaptability and valuable wood properties. The development and integration of genomic resources into breeding practice will be increasingly important in the decades to come. Bacterial artificial chromosome (BAC) libraries are key genomic tools that enable positional cloning of important traits, synteny evaluation, and the development of genome framework physical maps for genetic linkage and genome sequencing.ResultsWe describe the construction and characterization of two deep-coverage BAC libraries EG_Ba and EG_Bb obtained from nuclear DNA fragments of E. grandis (clone BRASUZ1) digested with HindIII and BstYI, respectively. Genome coverages of 17 and 15 haploid genome equivalents were estimated for EG_Ba and EG_Bb, respectively. Both libraries contained large inserts, with average sizes ranging from 135 Kb (Eg_Bb) to 157 Kb (Eg_Ba), very low extra-nuclear genome contamination providing a probability of finding a single copy gene ≥ 99.99%. Libraries were screened for the presence of several genes of interest via hybridizations to high-density BAC filters followed by PCR validation. Five selected BAC clones were sequenced and assembled using the Roche GS FLX technology providing the whole sequence of the E. grandis chloroplast genome, and complete genomic sequences of important lignin biosynthesis genes.ConclusionsThe two E. grandis BAC libraries described in this study represent an important milestone for the advancement of Eucalyptus genomics and forest tree research. These BAC resources have a highly redundant genome coverage (> 15×), contain large average inserts and have a very low percentage of clones with organellar DNA or empty vectors. These publicly available BAC libraries are thus suitable for a broad range of applications in genetic and genomic research in Eucalyptus and possibly in related species of Myrtaceae, including genome sequencing, gene isolation, functional and comparative genomics. Because they have been constructed using the same tree (E. grandis BRASUZ1) whose full genome is being sequenced, they should prove instrumental for assembly and gap filling of the upcoming Eucalyptus reference genome sequence.

Discovery of single‐nucleotide polymorphisms (SNPs) in the uncharacterized genome of the ascomycete Ophiognomonia clavigignenti‐juglandacearum from 454 sequence data

The benefits from recent improvement in sequencing technologies, such as the Roche GS FLX (454) pyrosequencing, may be even more valuable in non-model organisms, such as many plant pathogenic fungi of economic importance. One application of this new sequencing technology is the rapid generation of genomic information to identify putative single-nucleotide polymorphisms (SNPs) to be used for population genetic, evolutionary, and phylogeographic studies on non-model organisms. The focus of this research was to sequence, assemble, discover and validate SNPs in a fungal genome using 454 pyrosequencing when no reference sequence is available. Genomic DNA from eight isolates of Ophiognomonia clavigignenti-juglandacearum was pooled in one region of a four-region sequencing run on a Roche 454 GS FLX. This yielded 71 million total bases comprising 217,000 reads, 80% of which collapsed into 16,125,754 bases in 30,339 contigs upon assembly. By aligning reads from multiple isolates, we detected 298 SNPs using Roche's GS Mapper. With no reference sequence available, however, it was difficult to distinguish true polymorphisms from sequencing error. Eagleview software was used to manually examine each contig that contained one or more putative SNPs, enabling us to discard all but 45 of the original 298 putative SNPs. Of those 45 SNPs, 13 were validated using standard Sanger sequencing. This research provides a valuable genetic resource for research into the genus Ophiognomonia, demonstrates a framework for the rapid and cost-effective discovery of SNP markers in non-model organisms and should prove especially useful in the case of asexual or clonal fungi with limited genetic variability.

Comparative and Joint Analysis of Two Metagenomic Datasets from a Biogas Fermenter Obtained by 454-Pyrosequencing

Biogas production from renewable resources is attracting increased attention as an alternative energy source due to the limited availability of traditional fossil fuels. Many countries are promoting the use of alternative energy sources for sustainable energy production. In this study, a metagenome from a production-scale biogas fermenter was analysed employing Roche's GS FLX Titanium technology and compared to a previous dataset obtained from the same community DNA sample that was sequenced on the GS FLX platform. Taxonomic profiling based on 16S rRNA-specific sequences and an Environmental Gene Tag (EGT) analysis employing CARMA demonstrated that both approaches benefit from the longer read lengths obtained on the Titanium platform. Results confirmed Clostridia as the most prevalent taxonomic class, whereas species of the order Methanomicrobiales are dominant among methanogenic Archaea. However, the analyses also identified additional taxa that were missed by the previous study, including members of the genera Streptococcus, Acetivibrio, Garciella, Tissierella, and Gelria, which might also play a role in the fermentation process leading to the formation of methane. Taking advantage of the CARMA feature to correlate taxonomic information of sequences with their assigned functions, it appeared that Firmicutes, followed by Bacteroidetes and Proteobacteria, dominate within the functional context of polysaccharide degradation whereas Methanomicrobiales represent the most abundant taxonomic group responsible for methane production. Clostridia is the most important class involved in the reductive CoA pathway (Wood-Ljungdahl pathway) that is characteristic for acetogenesis. Based on binning of 16S rRNA-specific sequences allocated to the dominant genus Methanoculleus, it could be shown that this genus is represented by several different species. Phylogenetic analysis of these sequences placed them in close proximity to the hydrogenotrophic methanogen Methanoculleus bourgensis. While rarefaction analyses still indicate incomplete coverage, examination of the GS FLX Titanium dataset resulted in the identification of additional genera and functional elements, providing a far more complete coverage of the community involved in anaerobic fermentative pathways leading to methane formation.

Effective gene collection from the metatranscriptome of marine microorganisms

BackgroundMetagenomic studies, accelerated by the evolution of sequencing technologies and the rapid development of genomic analysis methods, can reveal genetic diversity and biodiversity in various samples including those of uncultured or unknown species. This approach, however, cannot be used to identify active functional genes under actual environmental conditions. Metatranscriptomics, which is similar in approach to metagenomics except that it utilizes RNA samples, is a powerful tool for the transcriptomic study of environmental samples. Unlike metagenomic studies, metatranscriptomic studies have not been popular to date due to problems with reliability, repeatability, redundancy and cost performance. Here, we propose a normalized metatranscriptomic method that is suitable for the collection of genes from samples as a platform for comparative transcriptomics.ResultsWe constructed two libraries, one non-normalized and the other normalized library, from samples of marine microorganisms taken during daylight hours from Hiroshima bay in Japan. We sequenced 0.6M reads for each sample on a Roche GS FLX, and obtained 0.2M genes after quality control and assembly. A comparison of the two libraries showed that the number of unique genes was larger in the normalized library than in the non-normalized library. Functional analysis of genes revealed that a small number of gene groups, ribosomal RNA genes and chloroplast genes, were dominant in both libraries. Taxonomic distribution analysis of the libraries suggests that Stramenopiles form a major taxon that includes diatoms. The normalization technique thus increases unique genes, functional categories of genes, and taxonomic richness.ConclusionsNormalization of the marine metatranscriptome could be useful in increasing the number of genes collected, and in reducing redundancies among highly expressed genes. Gene collection through the normalization method was effective in providing a foundation for comparative transcriptomic analysis.

Whole Kinome Profiling In Pediatric Acute Leukemia.

AbstractAbstract 1029With almost 130.000 entries in PubMed by today, the kinase family represents one of the gene families with the best documented history in malignant diseases.Acute myeloid leukemias account for 4.7% of all childhood cancers and 13.7% of all childhood leukemias. Recent german statistics (Kinderkrebsregister Mainz) indicate a 5-year survival rate of only 66%, emphasizing the need to discover novel therapeutic targets and prognostic markers. Here we present an adaptable workflow to detect mutations in kinase family members on genomic and expression level as well as in silico assessment of their functional consequences.To undertake the venture of resequencing the human kinome of leukemia patients, we constructed a custom SequenceCapture microarray (NimbleGen) based on all exons of all 518 members of the kinase family previously reported by Greenman and colleagues (Greenman et al, Nature, 2007) as well as a set of TaqMan® low density arrays (Life Technologies) to confirm the expression of potential candidates in tumor material. Genomic DNA from the initial tumor sample and germline material from a 16 year old female patient diagnosed with a AML FAB1 with Auer bodies, 93% blasts, CD33+ CD117+ CD38+ and partial CD15+ and from a 16 year old female patient with a bilinear CD22+ CD33+ leukemia was enriched on SequenceCapture arrays and sequenced on the Roche GS FLX Titanium platform. Acquired sequences were mapped against the current human reference (hg19/GRCh37). RNA derived from the initial leukemic blasts was used for kinome-wide expression profiling on TaqMan low density arrays.From sequencing the enriched DNA, we recieved between 85 and 129 megabases of tumor and germline sequence, of which 97.7–99.4% mapped to the human reference. About 90% of the targeted regions were covered at least once. Mapping and comparison to the germline material led to 28 and 46 tumor specific non-synonymous single nucleotide variants sequenced at a depth of ≥10x that were not previously reported as SNPs (dbSNP130). Analyzing these candidates with UniProt revealed that 6 and 12 were located in or very close to important functional domains of their respective protein. Structural protein models of one patients remaining mutations underscore the potential influence of detected mutations on the protein function. TaqMan® expression profiling of all kinases present on the SequenceCapture Array identified 3 and 4 of the remaining genes to be expressed in the tumor on mRNA level. In conclusion, starting from 518 candidates, we present a powerful workflow for high throughput analysis of the whole kinome in leukemic samples as well as CDK4, DYRK1B, GSG2 (pt1) and MASTL, MARK3, RP6KA5, CLK4 (pt2) as potential targets for further functional analysis. Disclosures:No relevant conflicts of interest to declare.

Optimization of whole-transcriptome amplification from low cell density deep-sea microbial samples for metatranscriptomic analysis

Limitation in sample quality and quantity is one of the big obstacles for applying metatranscriptomic technologies to explore gene expression and functionality of microbial communities in natural environments. In this study, several amplification methods were evaluated for whole-transcriptome amplification of deep-sea microbial samples, which are of low cell density and high impurity. The best amplification method was identified and incorporated into a complete protocol to isolate and amplify deep-sea microbial samples. In the protocol, total RNA was first isolated by a modified method combining Trizol (Invitrogen, CA) and RNeasy (QIAGEN, CA) method, amplified with a WT-Ovation™ Pico RNA Amplification System (NuGEN, CA), and then converted to double-strand DNA from single-strand cDNA with a WT-Ovation™ Exon Module (NuGEN, CA). The products from the whole-transcriptome amplification of deep-sea microbial samples were assessed first through random clone library sequencing. The BLAST search results showed that marine-based sequences are dominant in the libraries, consistent with the ecological source of the samples. The products were then used for next-generation Roche GS FLX Titanium sequencing to obtain metatranscriptome data. Preliminary analysis of the metatranscriptomic data showed good sequencing quality. Although the protocol was designed and demonstrated to be effective for deep-sea microbial samples, it should be applicable to similar samples from other extreme environments in exploring community structure and functionality of microbial communities.

Transcriptome Analysis ofTaxus cuspidataNeedles Based on 454 Pyrosequencing

Taxus species are highly valued as renewable resources for the production of Taxol. Despite the commercial and medicinal importance of Taxus, little genomic information is available for yew species, and Taxol biosynthesis still needs to be fully elucidated. In this study, 454 pyrosequencing technology was employed to produce an expressed sequence tag (EST) from the needles of Taxus cuspidata. In all, 81 148 high-quality reads from the needles of T. cuspidata were produced using Roche GS FLX Titanium. A total of 20,557 unique sequences were obtained, including 12 975 singletons and 7582 contigs. Approximately 14,095 unique sequences were annotated by a similarity search against five public databases. Gene ontology revealed 11,220 unique sequences that could be assigned to 45 vocabularies. In the Kyoto Encyclopedia of Genes and Genomes mapping, 2403 transcripts were established as associated with 3821 biochemical pathways. Enzymes in the plastidial 2-C-methyl-D-erythritol 4-phosphate pathway were well represented. Candidates of the putative genes of Taxol biosynthesis were revealed, including those in the remaining steps. In total, 291 transcripts were identified, representing putative homologues of transcription factors. Furthermore, 753 simple sequence repeat motifs, which are potential molecular markers for genetic application, were identified. These results provide the largest EST collections in TAXUS and will contribute to biosynthetic and biochemical studies that lead to drug improvement.

Depicting more accurate pictures of protistan community complexity using pyrosequencing of hypervariable SSU rRNA gene regions

Initial environmental pyrosequencing studies suggested highly complex protistan communities with phylotype richness decisively higher than previously estimated. However, recent studies on individual bacteria or artificial bacterial communities evidenced that pyrosequencing errors may skew our view of the true complexity of microbial communities. We pyrosequenced two diversity markers (hypervariable regions V4 and V9 of the small-subunit rDNA) of an intertidal protistan model community, using the Roche GS-FLX and the most recent GS-FLX Titanium sequencing systems. After pyrosequencing 24 reference sequences we obtained up to 2039 unique tags (from 3879 V4 GS-FLX Titanium reads), 77% of which were singletons. Even binning sequences that share 97% similarity still emulated a pseudodiversity exceeding the true complexity of the model community up to three times (V9 GS-FLX). Pyrosequencing error rates were higher for V4 fragments compared with the V9 domain and for the GS-FLX Titanium compared with the GS-FLX system. Furthermore, this experiment revealed that error rates are taxon-specific. As an outcome of this study we suggest a fast and efficient strategy to discriminate pyrosequencing signals from noise in order to more realistically depict the structure of protistan communities using simple tools that are implemented in standard tag data-processing pipelines.

Characterization of de novo transcriptome for waterhemp (Amaranthus tuberculatus) using GS‐FLX 454 pyrosequencing and its application for studies of herbicide target‐site genes

Waterhemp is a model for weed genomics research in part because it possesses many interesting biological characteristics, rapidly evolves resistance to herbicides and has a solid foundation of previous genetics work. To develop further the genomics resources for waterhemp, the transcriptome was sequenced using Roche GS-FLX 454 pyrosequencing technology. Pyrosequencing produced 483 225 raw reads, which, after quality control and assembly, yielded 44 469 unigenes (contigs + singletons). A total of 49% of these unigenes displayed highly significant similarities to Arabidopsis proteins and were subsequently grouped into gene ontology categories. Blast searches against public and custom databases helped in identifying and obtaining preliminary sequence data for all of the major target-site genes for which waterhemp has documented resistance. Moreover, sequence data for two other herbicide targets [4-hydroxyphenylpyruvate dioxygenase (HPPD) and glutamine synthetase], where resistance has not yet been reported in any plant, were also investigated in waterhemp and six related weedy Amaranthus species. These results demonstrate the enormous value of 454 sequencing for gene discovery and polymorphism detection in a major weed species and its relatives. Furthermore, the merging of the 454 transcriptome data with results from a previous whole genome 454 sequencing experiment has made it possible to establish a valuable genomic resource for weed science research.

Analysis of Multitag Pyrosequence Data from Human Cervical Lavage Samples

We have been using the Roche GS-FLX sequencing platform to produce tens of thousands of sequencing reads from samples of both bacterial communities (microbiome) and fungal communities (mycobiome) of stool, gut mucosa, vaginal washes, and oral washes from a large number of subjects. This vast volume of data from diverse sources has necessitated the development of an analysis pipeline in order to systematically and rapidly identify the taxa within the samples and to correlate the sample data with clinical and environmental features. Specifically, we have developed automated analytical tools for data tracking, taxonomical analysis, and feature clustering of bacteria in the human microbiome and demonstrate the pipeline using Cervical Vaginal Lavage (CVL) samples. This analysis pipeline will not only provide insight to our specific CVL dataset, but is applicable to other microbiome samples and will ultimately broaden our understanding of how the microbiome influences human health.

Abstract 4234: Translational regulation mediates hematopoietic progenitor cell generation in embryonic stem cell culture

Abstract The development and maintenance of hematopoietic progenitors (HP) requires modulation of a network of genes that balance self-renewal, expansion and lineage commitment. Regulation by miRNAs is critical for the differentiation of progenitors along defined pathways, and requires the recruitment and coordinate interaction of RNA binding proteins that modulate translation and/or target specific transcripts for sequestration or degradation. Here we provide evidence that translational regulation by the RNA binding proteins HuR and Pum2 are critical to HP generation from murine embryonic stem (ES) cell culture and that translational regulation during HP generation enhance ex vivo proliferative capacity and transplantability of progenitors. Translationally regulated transcript subsets in ES culture were identified by sucrose gradient polysome analysis. Ribonucleases and RNA-binding proteins comprised 12% of transcripts downregulated during ES cell differentiation to hematopoietic lineage (p=2.34 × 10-27 and 7.3 × 10-7 respectively). RNA-immunoprecipitation-sequencing (RIP-seq) using antibodies to several of the identified RNA binding proteins, followed by sequencing (Roche GS-FLX) revealed transcripts regulated by each. Pathway analysis of the transcripts associated with Pum2 in differentiated cells identified p53, p53-interacting proteins, and translation regulation Go (p=2.48×10-11). There was significant overlap with transcripts associated with HuR from undifferentiated ES (p=8.1×10-11), suggesting that the transcripts regulated by HuR in undifferentiated cells become associated with Pum2 with upon differentiation. Confocal imaging of HuR and Pum2 was consistent with this finding. Pathway analysis of DnD-associated transcripts revealed a network centered about Rac signaling and the Abl, Src and MAPK pathway via MAPKKK1–an essential pathway for stem cell self-renewal and maintenance. Consistent with the central role of translational regulation in the generation of HP, inhibition of translation using pharmacologic approaches (rapamycin, wortmannin, or clotrimazole) or siRNA (HuR, Pum2) resulted in up to three log increases in early HP derived from ES cell culture as measured by long term culture initiating cell (LTCIC) assay and the acquisition of competitive transplantability in irradiated hosts as measured by percentage chimeric animals and survival (p&amp;lt;0.05). Our findings reveal a system of broad-based translational regulation of HP differentiation mediated by RNA binding proteins. The findings suggest a methodology for ex vivo expansion of marrow-derived HP and enhanced generation of ES-derived HP by coordinate regulation of specific RNA binding proteins and their associated transcripts to regulate cell differentiation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4234.

Massively parallel sequencing of ataxia genes after array-based enrichment

Massively parallel sequencing has tremendous diagnostic potential but requires enriched templates for sequencing. Here we report the validation of an array-based sequence capture method in genetically heterogeneous disorders. The model disorder selected was AR ataxia, using five subjects with known mutations and two unaffected controls. The genomic sequences of seven disease genes, together with two control loci were targeted on a 2-Mb sequence-capture array. After enrichment, the patients' DNA samples were analyzed using one-quarter Roche GS FLX Titanium sequencing run, resulting in an average of 65 Mb of sequence data per patient. This was sufficient for an average 25-fold coverage/base in all targeted regions. Enrichment showed high specificity; on average, 80% of uniquely mapped reads were on target. Importantly, this approach enabled automated detection of deletions and hetero- and homozygous point mutations for 6/7 mutant alleles, and greater than 99% accuracy for known SNP variants. Our results also clearly show reduced coverage for sequences in repeat-rich regions, which significantly impacts the reliable detection of genomic variants. Based on these findings we recommend a minimal coverage of 15-fold for diagnostic implementation of this technology. We conclude that massive parallel sequencing of enriched samples enables personalized diagnosis of heterogeneous genetic disorders and qualifies for rapid diagnostic implementation.

Deep Sequencing the Tet2 Gene in 360 Patients with Myeloid Neoplasms Provides a Comprehensive and Quantitative Mutation Map and Reveals Low Level Mutant Clones.

Abstract 733Tet2 is a novel candidate tumor suppressor and mutations in this gene have been found in 24% of myeloid neoplasms following conventional Sanger sequencing. Using a massively parallel pyrosequencing methodology (Roche GS FLX) we sequenced the entire Tet2 gene coding region in 360 patients with myeloid malignancies (MDS 279; CMML 32; AML 10; MDS/MPD 10; 5q syndrome 8; others 21). We also utilized high density SNP6 arrays to screen for chromosomal aberrations including those at the Tet2 locus itself. Initially we found 106 mutations in Tet2 corresponding to 75 patients (21%), where the average sequence coverage was more than 100-fold for >90% of the gene. 25 patients had more than one mutant Tet2 clone. 83 mutations (in 17% of patients) were found in 30% to 95% of sequenced clones, where 50% or 100% could be indicative of heterozygous and homozygous mutations that would be found in all sample bone marrow cells respectively. The remaining 23 mutations were found in ≤30% of sequence clones, applying a lower threshold of 17% to avoid false positives, and were not reliably picked up by confirmatory Sanger sequencing. The 106 mutations were distributed as 44 nonsense or InsDel mutations throughout the gene, 59 nonsynonomous mutations within (23) and outside (36) the two known conserved domains, and 3 spice site mutations.Tet2 mutations were not correlated with survival outcome (probability of survival after 3 and 5 years from diagnosis 40% and 38% [p=0.3 log rank]for mutants vs non-mutants respectively). Patients with small (≤30%) or multiple mutant clones did not deviate from the overall survival trend. Furthermore, no difference in survival was seen when patients were analysed according to their WHO subgroups or IPSS scores. CMML featured as the only subgroup with a relatively high Tet2 mutation rate (53% of patients). Chromosomal aberrations linked to the Tet2 locus, including UPD or micro-deletion, were detected in 20-25% of patients, but this was not strongly correlated to detectable mutations in the Tet2 gene itself (24% coincidence), perhaps indicating that several forms of mutation or instability at this region are linked to disease. Interestingly, high percentage mutant clones which are indicative of homozygosity and thus loss of heterozygosity (LOH), were found as InsDel mutations in Tet2 for the majority (85%), suggesting a common defect in DNA replication or repair.Following up on additional smaller Tet2 mutant clones, we lowered our threshold for mutation detection and after further analysis and removal of experimental background, 42 additional candidate mutations of clone size between 8% and 17% were found. Taken as a whole, Tet2 gene mutations were found in 30% of patients but this may rise considerably if deeper sequencing screens for even smaller mutant clones yield further positive results.We therefore show via highly sensitive ‘Deep sequencing' on a relatively large patient cohort a comprehensive and highly quantitative mutation map across the Tet2 gene to allow increased prognostic comparisons. We also show that mutation figures rise significantly when smaller mutant clones are considered that cannot be detected via Sanger sequencing. In addition to this, if larger chromosomal aberrations such as UPD are added to the evolving number of mutant Tet2 patients, then Tet2 abnormalities may in fact be present in a majority of patients with myeloid neoplasms. Disclosures:Gattermann:Novartis: Honoraria, Participation in Advisory Boards on deferasirox clinical trials.

Direct multiplex sequencing (DMPS)—a novel method for targeted high-throughput sequencing of ancient and highly degraded DNA

Although the emergence of high-throughput sequencing technologies has enabled whole-genome sequencing from extinct organisms, little progress has been made in accelerating targeted sequencing from highly degraded DNA. Here, we present a novel and highly sensitive method for targeted sequencing of ancient and degraded DNA, which couples multiplex PCR directly with sample barcoding and high-throughput sequencing. Using this approach, we obtained a 96% complete mitochondrial genome data set from 31 cave bear (Ursus spelaeus) samples using only two 454 Life Sciences (Roche) GS FLX runs. In contrast to previous studies relying only on short sequence fragments, the overlapping portion of our data comprises almost 10 kb of replicated mitochondrial genome sequence, allowing for the unambiguous differentiation of three major cave bear clades. Our method opens up the opportunity to simultaneously generate many kilobases of overlapping sequence data from large sets of difficult samples, such as museum specimens, medical collections, or forensic samples. Embedded in our approach, we present a new protocol for the construction of barcoded sequencing libraries, which is compatible with all current high-throughput technologies and can be performed entirely in plate setup.

Analysis of Australian fur seal diet by pyrosequencing prey DNA in faeces

DNA-based techniques have proven useful for defining trophic links in a variety of ecosystems and recently developed sequencing technologies provide new opportunities for dietary studies. We investigated the diet of Australian fur seals (Arctocephalus pusillus doriferus) by pyrosequencing prey DNA from faeces collected at three breeding colonies across the seals' range. DNA from 270 faecal samples was amplified with four polymerase chain reaction primer sets and a blocking primer was used to limit amplification of fur seal DNA. Pooled amplicons from each colony were sequenced using the Roche GS-FLX platform, generating > 20,000 sequences. Software was developed to sort and group similar sequences. A total of 54 bony fish, 4 cartilaginous fish and 4 cephalopods were identified based on the most taxonomically informative amplicons sequenced (mitochondrial 16S). The prevalence of sequences from redbait (Emmelichthys nitidus) and jack mackerel (Trachurus declivis) confirm the importance of these species in the seals' diet. A third fish species, blue mackerel (Scomber australasicus), may be a more important prey species than previously recognised. There were major differences in the proportions of prey DNA recovered in faeces from different colonies, probably reflecting differences in prey availability. Parallel hard-part analysis identified largely the same main prey species as did the DNA-based technique, but with lower species diversity and no remains from cartilaginous prey. The pyrosequencing approach presented significantly expands the capabilities of DNA-based methods of dietary analysis and is suitable for large-scale diet investigations on a broad range of animals.