Genome Sequencing Strategies Research Articles

A cost-effective and universal strategy for complete prokaryotic genome sequencing proposed by computer simulation

A cost-effective and universal strategy for complete prokaryotic genome sequencing proposed by computer simulation

A cost-effective and universal strategy for complete prokaryotic genome sequencing proposed by computer simulation

Background: Pyrosequencing techniques allow scientists to perform prokaryotic genome sequencing to achieve the draft genomic sequences within a few days. However, the assemblies with shotgun sequencing are usually composed of hundreds of contigs. A further multiplex PCR procedure is needed to fill all the gaps and link contigs into complete chromosomal sequence, which is the basis for prokaryotic comparative genomic studies. In this article, we study various pyrosequencing strategies by simulated assembling from 100 prokaryotic genomes. Findings: Simulation study shows that a single end 454 Jr. run combined with a paired end 454 Jr. run (8 kb library) can produce: 1) ~90% of 100 assemblies with 99.99%; 4) average false gene duplication rate is < 0.7%; 5) average false gene loss rate is < 0.4%. Conclusions: A single end 454 Jr. run combined with a paired end 454 Jr. run (8 kb library) is a cost-effective way for prokaryotic whole genome sequencing. This strategy provides solution to produce high quality draft assemblies for most of prokaryotic organisms within days. Due to the small number of assembled scaffolds, the following multiplex PCR procedure (for gap filling) would be easy. As a result, large scale prokaryotic whole genome sequencing projects may be finished within weeks.

HIV-1 subtype C envelope characteristics associated with divergent rates of chronic disease progression

BackgroundHIV-1 envelope diversity remains a significant challenge for the development of an efficacious vaccine. The evolutionary forces that shape the diversity of envelope are incompletely understood. HIV-1 subtype C envelope in particular shows significant differences and unique characteristics compared to its subtype B counterpart. Here we applied the single genome sequencing strategy of plasma derived virus from a cohort of therapy naïve chronically infected individuals in order to study diversity, divergence patterns and envelope characteristics across the entire HIV-1 subtype C gp160 in 4 slow progressors and 4 progressors over an average of 19.5 months.ResultsSequence analysis indicated that intra-patient nucleotide diversity within the entire envelope was higher in slow progressors, but did not reach statistical significance (p = 0.07). However, intra-patient nucleotide diversity was significantly higher in slow progressors compared to progressors in the C2 (p = 0.0006), V3 (p = 0.01) and C3 (p = 0.005) regions. Increased amino acid length and fewer potential N-linked glycosylation sites (PNGs) were observed in the V1-V4 in slow progressors compared to progressors (p = 0.009 and p = 0.02 respectively). Similarly, gp41 in the progressors was significantly longer and had fewer PNGs compared to slow progressors (p = 0.02 and p = 0.02 respectively). Positive selection hotspots mapped mainly to V1, C3, V4, C4 and gp41 in slow progressors, whereas hotspots mapped mainly to gp41 in progressors. Signature consensus sequence differences between the groups occurred mainly in gp41.ConclusionsThese data suggest that separate regions of envelope are under differential selective forces, and that envelope evolution differs based on disease course. Differences between slow progressors and progressors may reflect differences in immunological pressure and immune evasion mechanisms. These data also indicate that the pattern of envelope evolution is an important correlate of disease progression in chronic HIV-1 subtype C infection.

A practical guide to fungal genome projects: strategy, technology, cost and completion

Since the publication of the first fungal genome in 1996, over 400 fungal genome projects have been launched, which makes fungi the most sequenced eukaryotic taxon. In this review, we sought to address the two major challenges associated with such projects, such as obtaining genomic sequence with high quality and attaching relevant biological information to the genomic sequence and features. Using the available literature and our own experiences in fungal genomics, we outline here the critical steps and quality control procedures for each stage of the genome project from library construction, sequencing to assembly and annotation, which will provide practical guidelines to optimize genome sequencing and annotation strategies. In addition, a brief overview of available comparative genomics and visualization tools is provided to assist investigators along the course of biological discovery. We also highlight how the success of a genomic project depends on close collaboration among experts from biological and computational sciences.

Genome assembly quality: Assessment and improvement using the neutral indel model

We describe a statistical and comparative-genomic approach for quantifying error rates of genome sequence assemblies. The method exploits not substitutions but the pattern of insertions and deletions (indels) in genome-scale alignments for closely related species. Using two- or three-way alignments, the approach estimates the amount of aligned sequence containing clusters of nucleotides that were wrongly inserted or deleted during sequencing or assembly. Thus, the method is well-suited to assessing fine-scale sequence quality within single assemblies, between different assemblies of a single set of reads, and between genome assemblies for different species. When applying this approach to four primate genome assemblies, we found that average gap error rates per base varied considerably, by up to sixfold. As expected, bacterial artificial chromosome (BAC) sequences contained lower, but still substantial, predicted numbers of errors, arguing for caution in regarding BACs as the epitome of genome fidelity. We then mapped short reads, at approximately 10-fold statistical coverage, from a Bornean orangutan onto the Sumatran orangutan genome assembly originally constructed from capillary reads. This resulted in a reduced gap error rate and a separation of error-prone from high-fidelity sequence. Over 5000 predicted indel errors in protein-coding sequence were corrected in a hybrid assembly. Our approach contributes a new fine-scale quality metric for assemblies that should facilitate development of improved genome sequencing and assembly strategies.

166th ENMC International Workshop on Collagen type VI-related Myopathies, 22–24 May 2009, Naarden, The Netherlands

Twenty-four clinicians, basic scientists and representatives of a patient advocacy organisation and pharmaceutical companies from 8 countries (Australia, England, France, Germany, Italy, Switzerland, Turkey, USA) gathered in Naarden, The Netherlands, to discuss the group of neuromuscular disorders caused by the deficiency of collagen type VI (ColVI), collectively termed ColVI-related myopathies. This meeting focused on three main areas: (1) the great heterogeneity observed at the genetic and clinical levels and the resulting complexity of the molecular diagnosis; (2) the pathophysiological mechanisms linking mutations in the COL6A genes to muscle pathology; (3) the current strategies for therapeutic interventions which have led to the design of upcoming clinical trials. Two sessions were also dedicated to existing and future databases, and to outcome measures.

A compartmentalized approach to the assembly of physical maps

BackgroundPhysical maps have been historically one of the cornerstones of genome sequencing and map-based cloning strategies. They also support marker assisted breeding and EST mapping. The problem of building a high quality physical map is computationally challenging due to unavoidable noise in the input fingerprint data.ResultsWe propose a novel compartmentalized method for the assembly of high quality physical maps from fingerprinted clones. The knowledge of genetic markers enables us to group clones into clusters so that clones in the same cluster are more likely to overlap. For each cluster of clones, a local physical map is first constructed using FingerPrinted Contigs (FPC). Then, all the individual maps are carefully merged into the final physical map. Experimental results on the genomes of rice and barley demonstrate that the compartmentalized assembly produces significantly more accurate maps, and that it can detect and isolate clones that would induce "chimeric" contigs if used in the final assembly.ConclusionThe software is available for download at

Cancer Genome Sequencing—An Interim Analysis

With the publishing of the first complete whole genome of a human cancer and its paired normal, we have passed a key milestone in the cancer genome sequencing strategy. The generation of such data will, thanks to technical advances, soon become commonplace. As a significant number of proof-of-concept studies have been published, it is important to analyze now the likely implications of these data and how this information might frame cancer research in the near future. The diversity of genes mutated within individual tumor types, the most striking feature of all studies reported to date, challenges gene-centric models of tumorigenesis. Although cancer genome sequencing will revolutionize certain aspects of personalized care, the value of these studies in facilitating the development of new therapies, their primary goal, seems less promising. Most significantly, however, the cancer genome sequencing strategy, as currently applied, fails to characterize the most relevant genomic features of cancer-the mutational heterogeneity within individual tumors.

Near Eastern Neolithic genetic input in a small oasis of the Egyptian Western Desert

The Egyptian Western Desert lies on an important geographic intersection between Africa and Asia. Genetic diversity of this region has been shaped, in part, by climatic changes in the Late Pleistocene and Holocene epochs marked by oscillating humid and arid periods. We present here a whole genome analysis of mitochondrial DNA (mtDNA) and high-resolution molecular analysis of nonrecombining Y-chromosomal (NRY) gene pools of a demographically small but autochthonous population from the Egyptian Western Desert oasis el-Hayez. Notwithstanding signs of expected genetic drift, we still found clear genetic evidence of a strong Near Eastern input that can be dated into the Neolithic. This is revealed by high frequencies and high internal variability of several mtDNA lineages from haplogroup T. The whole genome sequencing strategy and molecular dating allowed us to detect the accumulation of local mtDNA diversity to 5,138 +/- 3,633 YBP. Similarly, theY-chromosome gene pool reveals high frequencies of the Near Eastern J1 and the North African E1b1b1b lineages, both generally known to have expanded within North Africa during the Neolithic. These results provide another piece of evidence of the relatively young population history of North Africa.

Advancing full length genome sequencing for human RNA viral pathogens.

In the face of numerous emerging and re-emerging viral threats, large-scale genome sequencing efforts are underway to monitor viral evolution in real-time. To fully appreciate the mechanisms of viral adaptation and evolution, and to also develop reagents and resources for a better molecular diagnosis of emerging and re-emerging viral infections, there has been an increasing effort toward producing full length viral genome sequences. To date, high-throughput platforms have been developed using traditional Sanger-based sequencing and there are currently prospects to apply next generation sequencing methods to develop an ultra high-throughput strategy for viral genome sequencing and analysis.

Comparative analysis reveals signatures of differentiation amid genomic polymorphism in Lake Malawi cichlids

Low coverage survey sequencing shows that although Lake Malawi cichlids are phenotypically and behaviorally diverse, they appear genetically like a subdivided population.

Advanced resources for plant genomics: a BAC library specific for the short arm of wheat chromosome 1B

Common wheat (Triticum aestivum L., 2n = 6x = 42) is a polyploid species possessing one of the largest genomes among the cultivated crops (1C is approximately 17 000 Mb). The presence of three homoeologous genomes (A, B and D), and the prevalence of repetitive DNA make sequencing the wheat genome a daunting task. We have developed a novel 'chromosome arm-based' strategy for wheat genome sequencing to simplify this task; this relies on sub-genomic libraries of large DNA inserts. In this paper, we used a di-telosomic line of wheat to isolate six million copies of the short arm of chromosome 1B (1BS) by flow sorting. Chromosomal DNA was partially digested with HindIII and used to construct an arm-specific BAC library. The library consists of 65 280 clones with an average insert size of 82 kb. Almost half of the library (45%) has inserts larger than 100 kb, while 18% of the inserts range in size between 75 and 100 kb, and 37% are shorter than 75 kb. We estimated the chromosome arm coverage to be 14.5-fold, giving a 99.9% probability of identifying a clone corresponding to any sequence on the short arm of 1B. Each chromosome arm in wheat can be flow sorted from an appropriate cytogenetic stock, and we envisage that the availability of chromosome arm-specific BAC resources in wheat will greatly facilitate the development of ready-to-sequence physical maps and map-based gene cloning.

Reduced representation sequencing: A success in maize and a promise for other plant genomes

Plant, and particularly cereal genomes, are challenging to sequence due to their large size and high repetitive DNA content. Gene-enrichment strategies are alternative or complementary approaches to complete genome sequencing that yield, rapidly and inexpensively, useful sequence data from large and complex genomes. The maize genome is large (2.7 Gbp) and contains large amounts of conserved repetitive elements. Furthermore, the high allelic diversity found between maize inbred lines may necessitate sequencing several inbred lines in order to recover the maize "gene pool". Two gene-enrichment approaches, methylation filtration (MF) and high C(o)t (HC) sequencing have been tested in maize and their ability to sample the gene space has been examined. Combined with other genomic sequencing strategies, gene-enriched genomic sequencing is a practical way to examine the maize gene pool, to order and orient the genic sequences on the genome, and to enable investigation of gene content of other complex plant genomes.

Enrichment of Gene-Coding Sequences in Maize by Genome Filtration

Approximately 80% of the maize genome comprises highly repetitive sequences interspersed with single-copy, gene-rich sequences, and standard genome sequencing strategies are not readily adaptable to this type of genome. Methodologies that enrich for genic sequences might more rapidly generate useful results from complex genomes. Equivalent numbers of clones from maize selected by techniques called methylation filtering and High C0t selection were sequenced to generate approximately 200,000 reads (approximately 132 megabases), which were assembled into contigs. Combination of the two techniques resulted in a sixfold reduction in the effective genome size and a fourfold increase in the gene identification rate in comparison to a nonenriched library.

Electrophoretic Karyotype Analysis in Fungi

The resolution of chromosomal-sized DNAs by PFGE has many applications that include karyotyping, strain identification of similar species, characterization of transformed strains, building of linkage maps, and preparation of DNA for genomic analysis. Successful electrophoretic separation of chromosomes is an empiric process in which the initial concentration of intact chromosome-sized DNA and the optimization of electrophoretic parameters are the most important experimental variables. Nonetheless, inherent attributes of the genome architecture of certain species may thwart success. When a karyotype contains numerous chromosomes of the same size and/or many large (greater than 8 Mb) chromosomes, no amount of manipulation of the electrophoretic parameters will resolve individual chromosome bands using present technology. Further, fungi display a surprising amount of intraspecific variation in both chromosome number and size, making it difficult to establish a standard "reference" karyotype for many species. Although PFGE is not a panacea for bringing genetics to species that lack classical genetic systems, it often does provide a way for developing a molecular linkage map in the absence of a formal genetic system. It is far faster than parasexual analysis in the discovery of linkage relationships. For genomics projects, DNA can be recovered from pulsed field gels and used to prepare chromosome-specific libraries. Where whole genome sequencing strategies are used, chromosomes separated by PFGE provide an anchor for sequencing data. Electrophoretic karyotypes can be probed with anonymous pieces of DNA from bacterial artificial chromosome (BAC) contigs, thereby facilitating the building of physical maps. In conclusion, despite its shortcomings, the PFGE technique underlies much of our current understanding of the physical nature of the fungal genome.

A new approach to genome mapping and sequencing: slalom libraries.

We describe here an efficient strategy for simultaneous genome mapping and sequencing. The approach is based on physically oriented, overlapping restriction fragment libraries called slalom libraries. Slalom libraries combine features of general genomic, jumping and linking libraries. Slalom libraries can be adapted to different applications and two main types of slalom libraries are described in detail. This approach was used to map and sequence (with approximately 46% coverage) two human P1-derived artificial chromosome (PAC) clones, each of approximately 100 kb. This model experiment demonstrates the feasibility of the approach and shows that the efficiency (cost-effectiveness and speed) of existing mapping/sequencing methods could be improved at least 5-10-fold. Furthermore, since the efficiency of contig assembly in the slalom approach is virtually independent of length of sequence reads, even short sequences produced by rapid, high throughput sequencing techniques would suffice to complete a physical map and a sequence scan of a small genome.

Random PCR-based genome sequencing: a non-divide-and-conquer strategy.

We propose a genome sequencing strategy, which is neither divide-and-conquer (clone by clone) nor the shotgun approach. Random PCR-based and PCR relay sequencing constitute the basis of this novel strategy. Most of the genome is sequenced by the former process that requires only a set of non-specific primers and a template DNA. Random PCR-based sequencing reduces redundancy in sequencing by exploiting known sequence information. The number of primers required for random PCR was significantly diminished by using a combination of primers. The former process can be partially replaced by the shotgun method, if necessary. The gap-filling process can be effectively performed by way of PCR relay. The feasibility of this strategy was demonstrated using the Escherichia coli genome. This strategy enhances the global effort towards genome sequencing by being available through the Internet and by allowing the use of preexisting sequence data.

Analysis of Expressed Sequence Tags (ESTs) of the Parasitic ProtozoaEntamoeba histolytica

A directional cDNA library constructed from mRNA of the trophozoite ofEntamoeba histolyticaHM-1:IMSS strain was used for the generation of expressed sequence tags (ESTs). From 5′ ends of the distinct cDNA clones, 105 ESTs were obtained. Of these, 30 clones (29%) were previously knownE. histolyticagenes. Forty-five clones (42%) had matches with entries for other organisms in the databases. These newE. histolyticagenes constituted a broad range of transcripts distributed among cytoplasmic structural and regulatory proteins, enzymes, nuclear and other proteins, and proteins of unknown function. Thirty clones (29%) had no significant database matches and thus potentially representE. histolytica-specific genes. These data ofE. histolyticagenes identified by nucleotide sequencing indicate the value of the adoption of genome sequencing strategies for the rapid increase in knowledge of organisms causing dysentery and liver abscess.

A new strategy for genome sequencing.

Existing approaches to sequencing the human genome are based on the assumption that each region to be sequenced must first be mapped. But there is a simpler strategy in which any number of laboratories can cooperate.

Rice genome analysis by integration of sequencing and mapping data

The current strategy and recent results for rice genome mapping and sequencing are presented. The required resources are outlined, the integration of information in a multidisciplinary team advocated, and the essential role of bioinformatics discussed.