Sequencing of the Dutch elm disease fungus genome using the Roche/454 GS-FLX Titanium System in a comparison of multiple genomics core facilities.
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ABSTRACT:
SUBMITTER: Forgetta V
PROVIDER: S-EPMC3526337 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundThe rapid evolution of 454 GS-FLX sequencing technology has not been accompanied by a reassessment of the quality and accuracy of the sequences obtained. Current strategies for decision-making and error-correction are based on an initial analysis by Huse et al. in 2007, for the older GS20 system based on experimental sequences. We analyze here the quality of 454 sequencing data and identify factors playing a role in sequencing error, through the use of an extensive dataset for Roche control DNA fragments.ResultsWe obtained a mean error rate for 454 sequences of 1.07%. More importantly, the error rate is not randomly distributed; it occasionally rose to more than 50% in certain positions, and its distribution was linked to several experimental variables. The main factors related to error are the presence of homopolymers, position in the sequence, size of the sequence and spatial localization in PT plates for insertion and deletion errors. These factors can be described by considering seven variables. No single variable can account for the error rate distribution, but most of the variation is explained by the combination of all seven variables.ConclusionsThe pattern identified here calls for the use of internal controls and error-correcting base callers, to correct for errors, when available (e.g. when sequencing amplicons). For shotgun libraries, the use of both sequencing primers and deep coverage, combined with the use of random sequencing primer sites should partly compensate for even high error rates, although it may prove more difficult than previous thought to distinguish between low-frequency alleles and errors.
| S-EPMC3116506 | biostudies-literature
Project description:The use of next generation sequencing has expanded our view on whole mammalian methylome patterns. In particular, it provides a genome-wide insight of local DNA methylation diversity at single nucleotide level and enables the examination of single chromosome sequence sections at a sufficient statistical power. We describe a bisulfite-based sequence profiling pipeline, Bi-PROF, which is based on the 454 GS-FLX Titanium technology that allows to obtain up to one million sequence stretches at single base pair resolution without laborious subcloning. To illustrate the performance of the experimental workflow connected to a bioinformatics program pipeline (BiQ Analyzer HT) we present a test analysis set of 68 different epigenetic marker regions (amplicons) in five individual patient-derived xenograft tissue samples of colorectal cancer and one healthy colon epithelium sample as a control. After the 454 GS-FLX Titanium run, sequence read processing and sample decoding, the obtained alignments are quality controlled and statistically evaluated. Comprehensive methylation pattern interpretation (profiling) assessed by analyzing 10 (2)-10 (4) sequence reads per amplicon allows an unprecedented deep view on pattern formation and methylation marker heterogeneity in tissues concerned by complex diseases like cancer.
| S-EPMC3781196 | biostudies-literature
Project description:BackgroundMicrosatellites are markers of choice in population genetics and genomics, as they provide useful insight into patterns and processes as diverse as genome evolutionary dynamics and demographic processes. The acquisition of microsatellites through multiplex-enriched libraries and 454 GS-FLX Titanium pyrosequencing is a promising new tool for the isolation of new markers in unknown genomes. This approach can also be used to evaluate the extent to which microsatellite-enriched libraries are representative of the genome from which they were isolated. In this study, we deciphered potential discrepancies in microsatellite content recovery for two reference genomes (Apis mellifera and Danio rerio), selected on the basis of their extreme heterogeneity in terms of the proportions and distributions of microsatellites on chromosomes.ResultsThe A. mellifera genome, in particular, was found to be highly heterogeneous, due to extremely high rates of recombination, with hotspots, but the only bias consistently introduced into pyrosequenced multiplex-enriched libraries concerned sequence length, with the overrepresentation of sequences 160 to 320 bp in length. Other deviations from expected proportions or distributions of motifs on chromosomes were observed, but the significance and intensity of these deviations was mostly limited. Furthermore, no consistent adverse competition between multiplexed probes was observed during the motif enrichment phase.ConclusionsThis approach therefore appears to be a promising strategy for improving the development of microsatellites, as it introduces no major bias in terms of the proportions and distribution of microsatellites.
| S-EPMC3091709 | biostudies-literature
Project description:Next-generation sequencing (NGS) is widely used in biomedical research, but its adoption has been limited in molecular diagnostics. One application of NGS is the targeted resequencing of genes whose mutations lead to an overlapping clinical phenotype. This study evaluated the comparative performance of the Illumina Genome Analyzer and Roche 454 GS FLX for the resequencing of 16 genes associated with hypertrophic cardiomyopathy (HCM). Using a single human genomic DNA sample enriched by long-range PCR (LR-PCR), 40 GS FLX and 31 Genome Analyzer exon variants were identified using >or=30-fold read-coverage and >or=20% read-percentage selection criteria. Twenty-seven platform concordant variants were Sanger-confirmed. The discordant variants segregated into two categories: variants with read coverages >or=30 on one platform but <30-fold on the alternate platform and variants with read percentages >or=20% on one platform but <20% on the alternate platform. All variants with <30-fold coverage were Sanger-confirmed, suggesting that the coverage criterion of >or=30-fold is too stringent for variant discovery. The variants with <20% read percentage were identified as reference sequence based on Sanger sequencing. These variants were found in homopolymer tracts and short-read misalignments, specifically in genes with high identity. The results of the current study demonstrate the feasibility of combining LR-PCR with the Genome Analyzer or GS FLX for targeted resequencing of HCM-associated genes.
| S-EPMC2884316 | biostudies-literature
Project description:BackgroundLycoris aurea, also called Golden Magic Lily, is an ornamentally and medicinally important species of the Amaryllidaceae family. To date, the sequencing of its whole genome is unavailable as a non-model organism. Transcriptomic information is also scarce for this species. In this study, we performed de novo transcriptome sequencing to produce the first comprehensive expressed sequence tag (EST) dataset for L. aurea using high-throughput sequencing technology.Methodology and principal findingsTotal RNA was isolated from leaves with sodium nitroprusside (SNP), salicylic acid (SA), or methyl jasmonate (MeJA) treatment, stems, and flowers at the bud, blooming, and wilting stages. Equal quantities of RNA from each tissue and stage were pooled to construct a cDNA library. Using 454 pyrosequencing technology, a total of 937,990 high quality reads (308.63 Mb) with an average read length of 329 bp were generated. Clustering and assembly of these reads produced a non-redundant set of 141,111 unique sequences, comprising 24,604 contigs and 116,507 singletons. All of the unique sequences were involved in the biological process, cellular component and molecular function categories by GO analysis. Potential genes and their functions were predicted by KEGG pathway mapping and COG analysis. Based on our sequence analysis and published literatures, many putative genes involved in Amaryllidaceae alkaloids synthesis, including PAL, TYDC OMT, NMT, P450, and other potentially important candidate genes, were identified for the first time in this Lycoris. Furthermore, 6,386 SSRs and 18,107 high-confidence SNPs were identified in this EST dataset.ConclusionsThe transcriptome provides an invaluable new data for a functional genomics resource and future biological research in L. aurea. The molecular markers identified in this study will provide a material basis for future genetic linkage and quantitative trait loci analyses, and will provide useful information for functional genomic research in future.
| S-EPMC3621892 | biostudies-literature
Project description:BACKGROUND: Jatropha curcas L. is an important non-edible oilseed crop with promising future in biodiesel production. However, factors like oil yield, oil composition, toxic compounds in oil cake, pests and diseases limit its commercial potential. Well established genetic engineering methods using cloned genes could be used to address these limitations. Earlier, 10,983 unigenes from Sanger sequencing of ESTs, and 3,484 unique assembled transcripts from 454 pyrosequencing of uncloned cDNAs were reported. In order to expedite the process of gene discovery, we have undertaken 454 pyrosequencing of normalized cDNAs prepared from roots, mature leaves, flowers, developing seeds, and embryos of J. curcas. RESULTS: From 383,918 raw reads, we obtained 381,957 quality-filtered and trimmed reads that are suitable for the assembly of transcript sequences. De novo contig assembly of these reads generated 17,457 assembled transcripts (contigs) and 54,002 singletons. Average length of the assembled transcripts was 916 bp. About 30% of the transcripts were longer than 1000 bases, and the size of the longest transcript was 7,173 bases. BLASTX analysis revealed that 2,589 of these transcripts are full-length. The assembled transcripts were validated by RT-PCR analysis of 28 transcripts. The results showed that the transcripts were correctly assembled and represent actively expressed genes. KEGG pathway mapping showed that 2,320 transcripts are related to major biochemical pathways including the oil biosynthesis pathway. Overall, the current study reports 14,327 new assembled transcripts which included 2589 full-length transcripts and 27 transcripts that are directly involved in oil biosynthesis. CONCLUSION: The large number of transcripts reported in the current study together with existing ESTs and transcript sequences will serve as an invaluable genetic resource for crop improvement in jatropha. Sequence information of those genes that are involved in oil biosynthesis could be used for metabolic engineering of jatropha to increase oil content, and to modify oil composition.
| S-EPMC3087711 | biostudies-literature
Project description:Understanding microbial populations in hospital environments is crucial for improving human health. Hospital-acquired infections are an increasing problem in intensive care units (ICU). In this work we present an exploration of bacterial diversity at inanimate surfaces of the ICU wards of the University Hospital A Coruña (Spain), as an example of confined hospital environment subjected to selective pressure, taking the entrance hall of the hospital, an open and crowded environment, as reference. Surface swab samples were collected from both locations and recovered DNA used as template to amplify a hypervariable region of the bacterial 16S rRNA gene. Sequencing of the amplicons was performed at the Roche 454 Sequencing Center using GS-FLX Titanium procedures. Reads were pre-processed and clustered into OTUs (operational taxonomic units), which were further classified. A total of 16 canonical bacterial phyla were detected in both locations. Members of the phyla Firmicutes (mainly Staphylococcus and Streptococcus) and Actinobacteria (mainly Micrococcaceae, Corynebacteriaceae and Brevibacteriaceae) were over-represented in the ICU with respect to the Hall. The phyllum Proteobacteria was also well represented in the ICU, mainly by members of the families Enterobacteriaceae, Methylobacteriaceae and Sphingomonadaceae. In the Hall sample, the phyla Proteobacteria, Bacteroidetes, Deinococcus-Thermus and Cyanobacteria were over-represented with respect to the ICU. Over-representation of Proteobacteria was mainly due to the high abundance of Enterobacteriaceae members. The presented results demonstrate that bacterial diversity differs at the ICU and entrance hall locations. Reduced diversity detected at ICU, relative to the entrance hall, can be explained by its confined character and by the existence of antimicrobial selective pressure. This is the first study using deep sequencing techniques made in hospital wards showing substantial hospital microbial diversity.
| S-EPMC3430676 | biostudies-literature
Project description:BACKGROUND: Although mice have long served as an animal model for periodontitis, information on the composition of their indigenous oral microbiota is limited. The aim of the current study was to characterize mouse oral bacterial flora by applying extensive parallel pyrosequencing using the latest model pyrosequencer, a Roche/454 Genome Sequencer FLX Titanium. In addition, the effect of Toll-like receptor (TLR) 2 deficiency on oral microbiota was evaluated. RESULTS: Eight oral bacterial communities of wild-type (n = 4) and TLR2 knock-out (n = 4) C57BL/6 mice were characterized by analyzing 80,046 reads of 16S rRNA genes obtained by pyrosequencing. Excluding the PCR primers, the average length of each sequencing product was 443 bp. The average species richness of the murine oral bacterial communities was estimated to be about 200, but the communities were dominated by only two main phyla and several species. Therefore, the bacterial communities were relatively simple. The bacterial composition of the murine oral microbiota was significantly different from that of humans, and the lack of TLR2 had a negligible effect on the murine oral microbiota. CONCLUSION: Pyrosequencing using the Roche/454 FLX Titanium successfully characterized mouse oral bacterial communities. The relatively simple oral bacterial communities of mice were not affected by TLR2 deficiency. These findings will provide a basis for future studies on the role of periodontal pathogens in the murine model of periodontitis.
| S-EPMC2873484 | biostudies-literature
Project description:Swine vesicular disease virus (SVDV) is an enterovirus that is both genetically and antigenically closely related to human coxsackievirus B5 within the Picornaviridae family. SVDV is the causative agent of a highly contagious (though rarely fatal) vesicular disease in pigs. We report a rapid method that is suitable for sequencing the complete protein-encoding sequences of SVDV isolates in which the RNA is relatively intact. The approach couples a single PCR amplification reaction, using only a single PCR primer set to amplify the near-complete SVDV genome, with deep-sequencing using a small fraction of the capacity of a Roche GS FLX sequencing platform. Sequences were initially verified through one of two criteria; either a match between a de novo assembly and a reference mapping, or a match between all of five different reference mappings performed against a fixed set of starting reference genomes with significant genetic distances within the same species of viruses. All reference mappings used an iterative method to avoid bias. Further verification was achieved through phylogenetic analysis against published SVDV genomes and additional Enterovirus B sequences. This approach allows high confidence in the obtained consensus sequences, as well as provides sufficiently high and evenly dispersed sequence coverage to allow future studies of intra-host variation.
| S-EPMC4016283 | biostudies-literature
Project description:BackgroundDespite their species abundance and primary economic importance, genomic information about copepods is still limited. In particular, genomic resources are lacking for the copepod Calanus sinicus, which is a dominant species in the coastal waters of East Asia. In this study, we performed de novo transcriptome sequencing to produce a large number of expressed sequence tags for the copepod C. sinicus.ResultsCopepodid larvae and adults were used as the basic material for transcriptome sequencing. Using 454 pyrosequencing, a total of 1,470,799 reads were obtained, which were assembled into 56,809 high quality expressed sequence tags. Based on their sequence similarity to known proteins, about 14,000 different genes were identified, including members of all major conserved signaling pathways. Transcripts that were putatively involved with growth, lipid metabolism, molting, and diapause were also identified among these genes. Differentially expressed genes related to several processes were found in C. sinicus copepodid larvae and adults. We detected 284,154 single nucleotide polymorphisms (SNPs) that provide a resource for gene function studies.ConclusionOur data provide the most comprehensive transcriptome resource available for C. sinicus. This resource allowed us to identify genes associated with primary physiological processes and SNPs in coding regions, which facilitated the quantitative analysis of differential gene expression. These data should provide foundation for future genetic and genomic studies of this and related species.
| S-EPMC3646036 | biostudies-literature