Genome Shotgun Sequencing Research Articles

Development of genomic‐derived simple sequence repeat markers in Hevea brasiliensis from 454 genome shotgun sequences

With 2 figures and 2 tablesAbstractIn an attempt to develop additional microsatellite markers in rubber tree, we employed a 454 pyrosequencing technology to obtain genomic shotgun sequences and subsequently identified over 24 000 putative simple sequence repeats (SSRs). A total of 418 potential SSR loci were chosen for an empirical validation. Two hundred and twenty‐four primer pairs yielded successful amplification, and 90 SSR markers exhibited polymorphism among the 18 rubber tree accessions evaluated. The number of alleles per locus ranged from 2 to 7, with an average of 3.44 alleles per SSR. The gene diversity of individual microsatellite loci displayed a broad range of values from 0.104 to 0.901 with a mean of 0.612. The polymorphism information content also varied greatly from 0.099 to 0.893 with an average of 0.577. We also identified a set of five highly informative markers that were able to unequivocally distinguish 18 rubber tree genotypes examined in this study.

High throughput sequencing methods for microbiome profiling: application to food animal systems

Analysis of microbial communities using high throughput sequencing methods began in the mid 2000s permitting the production of 1000s to 10,000s of sequence reads per sample and megabases of data per sequence run. This then unprecedented depth of sequencing allowed, for the first time, the discovery of the 'rare biosphere' in environmental samples. The technology was quickly applied to studies in several human subjects. Perhaps these early studies served as a reminder that though the microbes that inhabit mammals are known to outnumber host cells by an order of magnitude or more, most of these are unknown members of our second genome, or microbiome (as coined by Joshua Lederberg), because of our inability to culture them. High throughput methods for microbial 16S ribosomal RNA gene and whole genome shotgun (WGS) sequencing have now begun to reveal the composition and identity of archaeal, bacterial and viral communities at many sites, in and on the human body. Surveys of the microbiota of food production animals have been published in the past few years and future studies should benefit from protocols and tools developed from large-scale human microbiome studies. Nevertheless, production animal-related resources, such as improved host genome assemblies and increased numbers and diversity of host-specific microbial reference genome sequences, will be needed to permit meaningful and robust analysis of 16S rDNA and WGS sequence data.

A shot in the genome: how accurately do shotgun 454 sequences represent a genome?

BackgroundNext generation sequencing (NGS) provides a valuable method to quickly obtain sequence information from non-model organisms at a genomic scale. In principle, if sequencing is not targeted for a genomic region or sequence type (e.g. coding region, microsatellites) NGS reads can be used as a genome snapshot and provide information on the different types of sequences in the genome. However, no study has ascertained if a typical 454 dataset of low coverage (1/4-1/8 of a PicoTiter plate leading to generally less than 0.1x of coverage) represents all parts of genomes equally.FindingsPartial genome shotgun sequencing of total DNA (without enrichment) on a 454 NGS platform was used to obtain reads of Apis mellifera (454 reads hereafter). These 454 reads were compared to the assembled chromosomes of this species in three different aspects: (i) dimer and trimer compositions, (ii) the distribution of mapped 454 sequences along the chromosomes and (iii) the numbers of different classes of microsatellites. Highly significant chi-square tests for all three types of analyses indicated that the 454 data is not a perfect random sample of the genome. Only the number of 454 reads mapped to each of the 16 chromosomes and the number of microsatellites pooled by motif (repeat unit) length was not significantly different from the expected values. However, a very strong correlation (correlation coefficients greater than 0.97) was observed between most of the 454 variables (the number of different dimers and trimers, the number of 454 reads mapped to each chromosome fragments of one Mb, the number of 454 reads mapped to each chromosome, the number of microsatellites of each class) and their corresponding genomic variables.ConclusionsThe results of chi square tests suggest that 454 shotgun reads cannot be regarded as a perfect representation of the genome especially if the comparison is done on a finer scale (e.g. chromosome fragments instead of whole chromosomes). However, the high correlation between 454 and genome variables tested indicate that a high proportion of the variability of 454 variables is explained by their genomic counterparts. Therefore, we conclude that using 454 data to obtain information on the genome is biologically meaningful.

Pyrosequencing-Based Comparative Genome Analysis of Vibrio vulnificus Environmental Isolates

Between 1996 and 2006, the US Centers for Disease Control reported that the only category of food-borne infections increasing in frequency were those caused by members of the genus Vibrio. The Gram-negative bacterium Vibrio vulnificus is a ubiquitous inhabitant of estuarine waters, and is the number one cause of seafood-related deaths in the US. Many V. vulnificus isolates have been studied, and it has been shown that two genetically distinct subtypes, distinguished by 16S rDNA and other gene polymorphisms, are associated predominantly with either environmental or clinical isolation. While local genetic differences between the subtypes have been probed, only the genomes of clinical isolates have so far been completely sequenced. In order to better understand V. vulnificus as an agent of disease and to identify the molecular components of its virulence mechanisms, we have completed whole genome shotgun sequencing of three diverse environmental genotypes using a pyrosequencing approach. V. vulnificus strain JY1305 was sequenced to a depth of 33×, and strains E64MW and JY1701 were sequenced to lesser depth, covering approximately 99.9% of each genome. We have performed a comparative analysis of these sequences against the previously published sequences of three V. vulnificus clinical isolates. We find that the genome of V. vulnificus is dynamic, with 1.27% of genes in the C-genotype genomes not found in the E- genotype genomes. We identified key genes that differentiate between the genomes of the clinical and environmental genotypes. 167 genes were found to be specifically associated with environmental genotypes and 278 genes with clinical genotypes. Genes specific to the clinical strains include components of sialic acid catabolism, mannitol fermentation, and a component of a Type IV secretory pathway VirB4, as well as several other genes with potential significance for human virulence. Genes specific to environmental strains included several that may have implications for the balance between self-preservation under stress and nutritional competence.

Comprehensive genomic characterization of squamous cell carcinoma of the lung.

7006 Background: A third of patients with non-small cell lung cancer are diagnosed with squamous cell carcinoma (SCC) histology. This report describes findings from the comprehensive genomic analyses of 178 SCC samples. Methods: The Cancer Genome Atlas (TCGA) is conducting DNA, RNA, and miRNA sequencing along with DNA copy number profiling, quantification of mRNA expression and promoter methylation on surgically resected samples from previously untreated patients with stage I-III SCC of the lung. Results: The demographics of 178 patients enrolled in the study: median age 68 years (range: 40-85); female 47 (26%) and history of tobacco smoking 171 (96%). Over 30 sites of significant somatic copy number alteration (SCNA) were identified. Exome sequencing of 178 lung SCC and matched normal samples revealed 13 significantly mutated genes with a False Discovery Rate (FDR) of <0.01 and high expression levels, including TP53, CDKN2A, PTEN, KEAP1, and NFE2L2. Apart from the near universal loss of TP53 and CDKN2A, alterations in the NFE2L2/KEAP1 and PI3K/AKT pathways were found in 35% and 43% of tumors analyzed. mRNA expression profiling revealed four distinct expression subtypes, each one enriched with distinct mutations and SCNAs - classical (37%): NFE2L2 and KEAP1 mutations, FGFR kinase alterations, increased global methylation and the highest rate of tobacco use; basal (24%): alterations in FGFR kinases; secretory (24%): PDGFRA alterations; primitive (15%): RB1 mutations. Rearrangements involving several known tumor suppressors were detected by whole genome shotgun sequencing of 20 tumor/normal pairs and confirmed by RNA sequencing including PTEN, RB1, NOTCH1, NF1 and CDKN2A. CDKN2A loss by one of several mechanisms (deletion, mutation, rearrangement with loss of function and methylation) was observed in 72% of specimens. Potential therapeutic targets for clinical trials with currently available drugs were identified in 127 patients (75%). Conclusions: SCC of the lung is a distinct molecular subtype of lung cancer potentially amenable to distinct molecularly targeted therapies.

Genotypic intraspecies heterogeneity ofEnterococcus italicus: data from dairy environments

The diversity of a collection of 19 Enterococcus italicus strains isolated from different dairy sources was explored using a molecular polyphasic approach, comprising random amplification of polymorphic DNA (RAPD-PCR), repetitive element PCR (REP-PCR), plasmid profiling and ribotyping. The data obtained showed a high-level of biodiversity, not always correlated to the niche of isolation. Particularly, REP-PCR with primer BOXA1R and plasmid profiling allowed the best discrimination at strain level. Exploiting the genome shotgun sequence of the type strain of the species, available in public database, genes related to insertion sequences present on enterococcal Pathogenic Islands (ISEf1, IS905), determinants related to virulence factors (codifying for hemolysin and cell wall surface proteins), exogenously DNA (conjugal transfer protein, replication plasmid protein, pheromone shutdown protein, phage integrase/recombinase) and penicillin binding proteins system were detected. The presence of most of these genes seemed a common genetic trait in the Enterococcus genus, sur gene (cell wall surface protein) was only detected in strains of E. italicus. To our knowledge, this is the first time that specific primers, with the expection of the species-specific probe targeted to 16S rRNA gene, have been designed for this species.

Genome shotgun sequencing and development of microsatellite markers for gerbera ( Gerbera hybrida H.) by 454 GS-FLX

The objective of this research was to develop and characterize microsatellite markers for gerbera. We used shotgun sequencing with Roche 454 GS-FLX Titanium technology to identify microsatellite loci in gerbera genomic DNA ( Gerbera hybrida ). The total length of non-redundant sequences obtained was 22,527,019 bp, which consisted of 3,085 contigs and 28,249 singletons. We assembled 61,958 reads into 3,085 contigs, of which 114 (3.70%) contained microsatellite repeats. The average G+C content was 39.3%. Functional annotation to known sequences yielded 14.7% unigenes in the ‘Raon’ cultivar. Analysis of the gerbera genome DNA (‘Raon’) general library showed that sequences of (AT), (AG), (AAG) and (AAT) repeats appeared most often, whereas (AC), (AAC) and (ACC) were the least frequent. Primer pairs were designed for 80 loci. Only eight primer pairs produced reproducible polymorphic bands in the 28 gerbera accessions analyzed. A total of 30 alleles were identified from the eight polymorphic SSR loci, with two to eight alleles per locus (average level of 3.75). These markers will be useful for investigating genetic diversity and differentiation in gerbera. Keywords: Genetic diversity, genomics, microsatellite isolation, pyrosequencing, SSRs. African Journal of Biotechnology Vol. 11(29), pp. 7388-7396, 10 April, 2012

Shotgun Genome Sequence of the Large Purple Photosynthetic Bacterium Rhodospirillum photometricum DSM122

Here, we present the shotgun genome sequence of the purple photosynthetic bacterium Rhodospirillum photometricum DSM122. The photosynthetic apparatus of this bacterium has been particularly well studied by microscopy. The knowledge of the genome of this oversize bacterium will allow us to compare it with the other purple bacterial organisms to follow the evolution of the photosynthetic apparatus.

Single Molecule Sequencing of Free DNA from Maternal Plasma for Noninvasive Trisomy 21 Detection

Noninvasive fetal aneuploidy detection by use of free DNA from maternal plasma has recently been shown to be achievable by whole genome shotgun sequencing. The high-throughput next-generation sequencing platforms previously tested use a PCR step during sample preparation, which results in amplification bias in GC-rich areas of the human genome. To eliminate this bias, and thereby experimental noise, we have used single molecule sequencing as an alternative method. For noninvasive trisomy 21 detection, we performed single molecule sequencing on the Helicos platform using free DNA isolated from maternal plasma from 9 weeks of gestation onwards. Relative sequence tag density ratios were calculated and results were directly compared to the previously described Illumina GAII platform. Sequence data generated without an amplification step show no GC bias. Therefore, with the use of single molecule sequencing all trisomy 21 fetuses could be distinguished more clearly from euploid fetuses. This study shows for the first time that single molecule sequencing is an attractive and easy to use alternative for reliable noninvasive fetal aneuploidy detection in diagnostics. With this approach, previously described experimental noise associated with PCR amplification, such as GC bias, can be overcome.

Whole genome shotgun sequencing of one Colombian clinical isolate of Mycobacterium tuberculosis reveals DosR regulon gene deletions

Several genomes of different Mycobacterium tuberculosis isolates have been completely sequenced around the world. The genomic information obtained have shown higher diversity than originally thought and specific adaptations to different human populations. Within this work, we sequenced the genome of one Colombian M.tuberculosis virulent isolate. Genomic comparison against the reference genome of H37Rv and other strains showed multiple deletion and insertions that ranged between a few bases to thousands. Excluding PPE and PG-PGRS genes, 430 proteins present changes in at least 1 amino acid. Also, novel positions of the IS6110 mobile element were identified. This isolate is also characterized by a large genomic deletion of 3.6kb, leading to the loss and modification of the dosR regulon genes, Rv1996 and Rv1997. To our knowledge, this is the first report of the genome sequence of a Latin American M.tuberculosis clinical isolate.

To hype, or not to(o) hype

Scientists and journalists try to engage the public with exciting stories, but who is guilty of overselling research and what are the consequences?

Single Nucleotide Polymorphisms Detected and In Silico Analysis of the 5’ Flanking Sequence and Exon 1 in the Bubalus bubalis Leptin Gene

The leptin plays a critical role in the regulation of reproductive and immune function in humans, it is at the centre of the complex networks that coordinate changes in nutritional state with many diverse aspects of mammalian biology. In this study, we have sequenced the 5’ flanking region and exon 1 of the leptin gene in buffalo, and have detected eight single nucleotide polymorphisms; we have made evidence, through in silico analysis that many of them fall within putative binding sites for transcription factors. Starting from the bovine whole genome shotgun sequence, that encodes the complete sequence of the leptin gene, we had designed primers to amplify two amplicons, so to cover the 5’ flanking and exon 1 of the leptin gene of 41 non related buffaloes. The newly sequenced buffalo fragment was submitted to profile search for transcription factor binding sites, using the MATCHTM program, focusing on the areas where the single nucleotide polymorphisms had been detected. Our analysis shows that the majority of the identified single nucleotide polymorphisms fall into the core sequence of transcription factor binding sites that regulate the expression of target genes in many physiological processes within mammalian tissues. Because the leptin gene plays an important role in influencing economic traits in cattle, the novel detected single nucleotide polymorphisms might be used in association studies to assess their potential of being genetic markers for selection.

Genome-wide prediction of interferon family members of tree shrew and their molecular characteristics analysis

Interferons (IFNs) represent proteins with antiviral activities that are secreted from cells in response to a variety of stimuli. In addition to antiviral, antibacterial and anti-parasitic host-defense functions they are now also recognized as crucial regulators of cell proliferation, differentiation, survival and death as well as activators of specialized cell functions particularly in the immune system and play important roles in infectious and inflammatory diseases, autoimmunity and cancer. Tree shrews (Tupaia belangeri) were found to be susceptible to several human viruses and therefore are widely regarded as good models for analyzing mechanism of human diseases. In this report, we have forecasted the interferon family members of tree shrew from its genome mainly using the methods like Blast (whole genome shotgun sequence) and gene prediction. Our data show that tree shrew interferon system includes: type I IFN: α (five subtypes), β, ω, κ, epsilon, δ; type II IFN: γ; type III IFN: λ1, λ2/3. Furthermore, the predicted structures of α and λ have similar character with those of other mammals. However, there are some differences in cysteine position and N-glycosylation numbers between human and Tree shrew IFNs. These results provide fundamental basis for further molecular cloning and function analysis of tree shrew IFNs in future.

Comparative genomics of Neisseria weaveri clarifies the taxonomy of this species and identifies genetic determinants that may be associated with virulence

A group of bacterial strains formerly known as CDC group M-5 are opportunistic pathogens to humans. In 1993, a name, Neisseria weaveri, was proposed by two independent studies to harbor CDC group M-5 strains, namely N.weaveri Holmes etal. 1993 and N.weaveri Andersen etal. 1993, with two different 'type' strains. However, no study has been conducted on to the relatedness of the two 'type' strains, although the close relationship of the two taxa has long been accepted unofficially. Formally, the status of the name N.weaveri Andersen etal. 1993 is illegitimate because it is a later homonym of N.weaveri Holmes etal., 1993; but the name of the strain is still validly published. In this study, we attempt to resolve the confusion caused by the apparent duplication of the species N.weaveri (with different type strains) using whole genome shotgun sequencing. We also sought to gain insight into the genetic characteristics of N.weaveri by conducting comparative genomics. On the basis of genomic similarities revealed through a comparative genomic study, we propose that N.weaveri Andersen etal. 1993 should be re-classified as a later heterotypic synonym of N.weaveri Holmes etal., 1993.

The Cassava Genome: Current Progress, Future Directions

The starchy swollen roots of cassava provide an essential food source for nearly a billion people, as well as possibilities for bioenergy, yet improvements to nutritional content and resistance to threatening diseases are currently impeded. A 454-based whole genome shotgun sequence has been assembled, which covers 69% of the predicted genome size and 96% of protein-coding gene space, with genome finishing underway. The predicted 30,666 genes and 3,485 alternate splice forms are supported by 1.4 M expressed sequence tags (ESTs). Maps based on simple sequence repeat (SSR)-, and EST-derived single nucleotide polymorphisms (SNPs) already exist. Thanks to the genome sequence, a high-density linkage map is currently being developed from a cross between two diverse cassava cultivars: one susceptible to cassava brown streak disease; the other resistant. An efficient genotyping-by-sequencing (GBS) approach is being developed to catalog SNPs both within the mapping population and among diverse African farmer-preferred varieties of cassava. These resources will accelerate marker-assisted breeding programs, allowing improvements in disease-resistance and nutrition, and will help us understand the genetic basis for disease resistance.

Analysis of the salivary microbiome using culture-independent techniques

BackgroundThe salivary microbiota is a potential diagnostic indicator of several diseases. Culture-independent techniques are required to study the salivary microbial community since many of its members have not been cultivated.MethodsWe explored the bacterial community composition in the saliva sample using metagenomic whole genome shotgun (WGS) sequencing, the extraction of 16S rRNA gene fragments from metagenomic sequences (16S-WGS) and high-throughput sequencing of PCR-amplified bacterial 16S rDNA gene (16S-HTS) regions V1 and V3.ResultsThe hierarchical clustering of data based on the relative abundance of bacterial genera revealed that distances between 16S-HTS datasets for V1 and V3 regions were greater than those obtained for the same V region with different numbers of PCR cycles. Datasets generated by 16S-HTS and 16S-WGS were even more distant. Finally, comparison of WGS and 16S-based datasets revealed the highest dissimilarity.The analysis of the 16S-HTS, WGS and 16S-WGS datasets revealed 206, 56 and 39 bacterial genera, respectively, 124 of which have not been previously identified in salivary microbiomes. A large fraction of DNA extracted from saliva corresponded to human DNA. Based on sequence similarity search against completely sequenced genomes, bacterial and viral sequences represented 0.73% and 0.0036% of the salivary metagenome, respectively. Several sequence reads were identified as parts of the human herpesvirus 7.ConclusionsAnalysis of the salivary metagenome may have implications in diagnostics e.g. in detection of microorganisms and viruses without designing specific tests for each pathogen.

Derivation of HLA types from shotgun sequence datasets.

The human leukocyte antigen (HLA) is key to many aspects of human physiology and medicine. All current sequence-based HLA typing methodologies are targeted approaches requiring the amplification of specific HLA gene segments. Whole genome, exome and transcriptome shotgun sequencing can generate prodigious data but due to the complexity of HLA loci these data have not been immediately informative regarding HLA genotype. We describe HLAminer, a computational method for identifying HLA alleles directly from shotgun sequence datasets (http://www.bcgsc.ca/platform/bioinfo/software/hlaminer). This approach circumvents the additional time and cost of generating HLA-specific data and capitalizes on the increasing accessibility and affordability of massively parallel sequencing.

Analysis of a unique Clostridium botulinum strain from the Southern hemisphere producing a novel type E botulinum neurotoxin subtype

BackgroundClostridium botulinum strains that produce botulinum neurotoxin type E (BoNT/E) are most commonly isolated from botulism cases, marine environments, and animals in regions of high latitude in the Northern hemisphere. A strain of C. botulinum type E (CDC66177) was isolated from soil in Chubut, Argentina. Previous studies showed that the amino acid sequences of BoNT/E produced by various strains differ by < 6% and that the type E neurotoxin gene cluster inserts into the rarA operon.ResultsGenetic and mass spectral analysis demonstrated that the BoNT/E produced by CDC66177 is a novel toxin subtype (E9). Toxin gene sequencing indicated that BoNT/E9 differed by nearly 11% at the amino acid level compared to BoNT/E1. Mass spectrometric analysis of BoNT/E9 revealed that its endopeptidase substrate cleavage site was identical to other BoNT/E subtypes. Further analysis of this strain demonstrated that its 16S rRNA sequence clustered with other Group II C. botulinum (producing BoNT types B, E, and F) strains. Genomic DNA isolated from strain CDC66177 hybridized with fewer probes using a Group II C. botulinum subtyping microarray compared to other type E strains examined. Whole genome shotgun sequencing of strain CDC66177 revealed that while the toxin gene cluster inserted into the rarA operon similar to other type E strains, its overall genome content shared greater similarity with a Group II C. botulinum type B strain (17B).ConclusionsThese results expand our understanding of the global distribution of C. botulinum type E strains and suggest that the type E toxin gene cluster may be able to insert into C. botulinum strains with a more diverse genetic background than previously recognized.

Shotgun Genome Sequence of a Yersinia enterocolitica Isolate from the Philippines

The first shotgun genome sequence of a microbial pathogen from the Philippines is reported. Yersinia enterocolitica subsp. palearctica strain PhRBD_Ye1 is the first Y. enterocolitica strain sequenced from an animal source, swine, which is a natural source of yersiniosis. The closest phylogenetic match is a human clinical isolate from Germany.

MICROSATELLITE DEVELOPMENT IN RHODOPHYTA USING HIGH-THROUGHPUT SEQUENCE DATA(1).

Shotgun genome sequencing is rapidly emerging as the method of choice for the identification of microsatellite loci in nonmodel organisms. However, to the best of our knowledge, this approach has not been applied to marine algae so far. Herein, we report the results of using the 454 next-generation sequencing (NGS) platform to randomly sample 36.0 and 40.9 Mbp (139,786 and 139,795 reads, respectively) of the genome of two red algae from the northwest Iberian Peninsula [Grateloupia lanceola (J. Agardh) J. Agardh and a still undescribed new member of the family Cruoriaceae]. Using data mining tools, we identified 4,766 and 5,174 perfect microsatellite loci in 4,344 and 4,504 sequences/contigs from G. lanceola and the Cruoriaceae, respectively. After conservative removal of potentially problematic loci (redundant sequences, mobile elements), primer design was possible for 1,371 and 1,366 loci, respectively. A survey of the literature indicates that microsatellite density in our Rhodophyta is at the low end of the values reported for other organisms investigated with the same technology (land plants and animals). A limited number of loci were successfully tested for PCR amplification and polymorphism finding that they may be suitable for population genetic studies. This study demonstrates that random genome sequencing is a rapid, effective alternative to develop useful microsatellite loci in previously unstudied red algae.