Extension of Lander-Waterman theory for sequencing filtered DNA libraries

BACKGROUND: The degree to which conventional DNA sequencing techniques will be successful for highly repetitive genomes is unclear. Investigators are therefore considering various filtering methods to select against high-copy sequence in DNA clone libraries. The standard model for random sequencing, Lander-Waterman theory, does not account for two important issues in such libraries, discontinuities and position-based sampling biases (the so-called "edge effect"). We report an extension of the theory for analyzing such configurations. RESULTS: The edge effect cannot be neglected in most cases. Specifically, rates of coverage and gap reduction are appreciably lower than those for conventional libraries, as predicted by standard theory. Performance decreases as read length increases relative to island size. Although opposite of what happens in a conventional library, this apparent paradox is readily explained in terms of the edge effect. The model agrees well with prototype gene-tagging experiments for Zea mays and Sorghum bicolor. Moreover, the associated density function suggests well-defined probabilistic milestones for the number of reads necessary to capture a given fraction of the gene space. An exception for applying standard theory arises if sequence redundancy is less than about 1-fold. Here, evolution of the random quantities is independent of library gaps and edge effects. This observation effectively validates the practice of using standard theory to estimate the genic enrichment of a library based on light shotgun sequencing. CONCLUSION: Coverage performance using a filtered library is significantly lower than that for an equivalent-sized conventional library, suggesting that directed methods may be more critical for the former. The proposed model should be useful for analyzing future projects

Novel and nodulation-regulated microRNAs in soybean roots

<p>Abstract</p> <p>Background</p> <p>Small RNAs regulate a number of developmental processes in plants and animals. However, the role of small RNAs in legume-rhizobial symbiosis is largely unexplored. Symbiosis between legumes (e.g. soybean) and rhizobia bacteria (e.g. <it>Bradyrhizobium japonicum</it>) results in root nodules where the majority of biological nitrogen fixation occurs. We sought to identify microRNAs (miRNAs) regulated during soybean-<it>B. japonicum </it>symbiosis.</p> <p>Results</p> <p>We sequenced ~350000 small RNAs from soybean roots inoculated with <it>B. japonicum </it>and identified conserved miRNAs based on similarity to miRNAs known in other plant species and new miRNAs based on potential hairpin-forming precursors within soybean EST and shotgun genomic sequences. These bioinformatics analyses identified 55 families of miRNAs of which 35 were novel. A subset of these miRNAs were validated by Northern analysis and miRNAs differentially responding to <it>B. japonicum </it>inoculation were identified. We also identified putative target genes of the identified miRNAs and verified <it>in vivo </it>cleavage of a subset of these targets by 5'-RACE analysis. Using conserved miRNAs as internal control, we estimated that our analysis identified ~50% of miRNAs in soybean roots.</p> <p>Conclusion</p> <p>Construction and analysis of a small RNA library led to the identification of 20 conserved and 35 novel miRNA families in soybean. The availability of complete and assembled genome sequence information will enable identification of many other miRNAs. The conserved miRNA loci and novel miRNAs identified in this study enable investigation of the role of miRNAs in rhizobial symbiosis.</p

The C-Fern (Ceratopteris richardii) Genome: Insights Into Plant Genome Evolution With the First Partial Homosporous Fern Genome Assembly

Ferns are notorious for possessing large genomes and numerous chromosomes. Despite decades of speculation, the processes underlying the expansive genomes of ferns are unclear, largely due to the absence of a sequenced homosporous fern genome. The lack of this crucial resource has not only hindered investigations of evolutionary processes responsible for the unusual genome characteristics of homosporous ferns, but also impeded synthesis of genome evolution across land plants. Here, we used the model fern species Ceratopteris richardii to address the processes (e.g., polyploidy, spread of repeat elements) by which the large genomes and high chromosome numbers typical of homosporous ferns may have evolved and have been maintained. We directly compared repeat compositions in species spanning the green plant tree of life and a diversity of genome sizes, as well as both short- and long-read-based assemblies of Ceratopteris. We found evidence consistent with a single ancient polyploidy event in the evolutionary history of Ceratopteris based on both genomic and cytogenetic data, and on repeat proportions similar to those found in large flowering plant genomes. This study provides a major stepping-stone in the understanding of land plant evolutionary genomics by providing the first homosporous fern reference genome, as well as insights into the processes underlying the formation of these massive genomes

Advances in Genome Biology & Technology

This year's meeting focused on the latest advances in new DNA sequencing technologies and the applications of genomics to disease areas in biology and biomedicine. Daytime plenary sessions highlighted cutting-edge research in areas such as complex genetic diseases, comparative genomics, medical sequencing, massively parallel DNA sequencing, and synthetic biology. Technical approaches being developed and utilized in contemporary genomics research were presented during evening concurrent sessions. Also, as in previous years, poster sessions bridged the morning and afternoon plenary sessions. In addition, for the third year in a row, the Advances in Genome Biology and Technology (AGBT) meeting was preceded by a pre-meeting workshop that aimed to provide an introductory overview for trainees and other meeting attendees. This year, speakers at the workshop focused on next-generation sequencing technologies, including their experiences, findings, and helpful advise for others contemplating using these platforms in their research. Speakers from genome centers and core sequencing facilities were featured and the workshop ended with a roundtable discussion, during which speakers fielded questions from the audience

Optical mapping as a routine tool for bacterial genome sequence finishing

Background: In sequencing the genomes of two Xenorhabdus species, we encountered a large number of sequence repeats and assembly anomalies that stalled finishing efforts. This included a stretch of about 12 Kb that is over 99.9% identical between the plasmid and chromosome of X. nematophila. Results: Whole genome restriction maps of the sequenced strains were produced through optical mapping technology. These maps allowed rapid resolution of sequence assembly problems, permitted closing of the genome, and allowed correction of a large inversion in a genome assembly that we had considered finished. Conclusion: Our experience suggests that routine use of optical mapping in bacterial genome sequence finishing is warranted. When combined with data produced through 454 sequencing, an optical map can rapidly and inexpensively generate an ordered and oriented set of contigs to produce a nearly complete genome sequence assembly

Deep expression analysis reveals distinct cold-response strategies in rubber tree (hevea brasiliensis)

Natural rubber, an indispensable commodity used in approximately 40,000 products, is fundamental to the tire industry. The rubber tree species Hevea brasiliensis (Willd. ex Adr. de Juss.) Muell-Arg., which is native the Amazon rainforest, is the major producer of latex worldwide. Rubber tree breeding is time consuming, expensive and requires large field areas. Thus, genetic studies could optimize field evaluations, thereby reducing the time and area required for these experiments. In this work, transcriptome sequencing was used to identify a full set of transcripts and to evaluate the gene expression involved in the different cold-response strategies of the RRIM600 (cold-resistant) and GT1 (cold-tolerant) genotypes.ResultsWe built a comprehensive transcriptome using multiple database sources, which resulted in 104,738 transcripts clustered in 49,304 genes. The RNA-seq data from the leaf tissues sampled at four different times for each genotype were used to perform a gene-level expression analysis. Differentially expressed genes (DEGs) were identified through pairwise comparisons between the two genotypes for each time series of cold treatments.DEG annotation revealed that RRIM600 and GT1 exhibit different chilling tolerance strategies. To cope with cold stress, the RRIM600 clone upregulates genes promoting stomata closure, photosynthesis inhibition and a more efficient reactive oxygen species (ROS) scavenging system. The transcriptome was also searched for putative molecular markers (single nucleotide polymorphisms (SNPs) and microsatellites) in each genotype. and a total of 27,111 microsatellites and 202,949 (GT1) and 156,395 (RRIM600) SNPs were identified in GT1 and RRIM600. Furthermore, a search for alternative splicing (AS) events identified a total of 20,279 events.ConclusionsThe elucidation of genes involved in different chilling tolerance strategies associated with molecular markers and information regarding AS events provides a powerful tool for further genetic and genomic analyses of rubber tree breeding20CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP478701/2012–8; 402954/2012Sem informação2007/50392–1; 2012/50491–8; 2014/18755–0; 2015/24346–

Transcriptomic Shock Generates Evolutionary Novelty in a Newly Formed, Natural Allopolyploid Plant

SummaryNew hybrid species might be expected to show patterns of gene expression intermediate to those shown by parental species [1, 2]. “Transcriptomic shock” may also occur, in which gene expression is disrupted; this may be further modified by whole genome duplication (causing allopolyploidy) [3–16]. “Shock” can include instantaneous partitioning of gene expression between parental copies of genes among tissues [16–19]. These effects have not previously been studied at a population level in a natural allopolyploid plant species. Here, we survey tissue-specific expression of 144 duplicated gene pairs derived from different parental species (homeologs) in two natural populations of 40-generation-old allotetraploid Tragopogon miscellus (Asteraceae) plants. We compare these results with patterns of allelic expression in both in vitro “hybrids” and hand-crossed F1 hybrids between the parental diploids T. dubius and T. pratensis, and with patterns of homeolog expression in synthetic (S1) allotetraploids. Partitioning of expression was frequent in natural allopolyploids, but F1 hybrids and S1 allopolyploids showed less partitioning of expression than the natural allopolyploids and the in vitro “hybrids” of diploid parents. Our results suggest that regulation of gene expression is relaxed in a concerted manner upon hybridization, and new patterns of partitioned expression subsequently emerge over the generations following allopolyploidization

Validation of reference transcripts in strawberry (<i>Fragaria</i> spp.)

Contemporary methods to assay gene expression depend on a stable set of reference transcripts for accurate quantitation. A lack of well-tested reference genes slows progress in characterizing gene expression in high-value specialty crops. In this study, a set of strawberry (Fragaria spp.) constitutively expressed reference genes has been identified by merging digital gene expression data with expression profiling. Constitutive reference candidates were validated using quantitative PCR and hybridization. Several transcripts have been identified that show improved stability across tissues relative to traditional reference transcripts. Results are similar between commercial octoploid strawberry and the diploid model. Our findings also show that while some never-before-used references are appropriate for most applications, even the most stable reference transcripts require careful assessment across the diverse tissues and fruit developmental states before being adopted as controls.Facultad de Ciencias ExactasInstituto de Fisiología Vegeta

The TIGR Maize Database

Maize is a staple crop of the grass family and also an excellent model for plant genetics. Owing to the large size and repetitiveness of its genome, we previously investigated two approaches to accelerate gene discovery and genome analysis in maize: methylation filtration and high C(0)t selection. These techniques allow the construction of gene-enriched genomic libraries by minimizing repeat sequences due to either their methylation status or their copy number, yielding a 7-fold enrichment in genic sequences relative to a random genomic library. Approximately 900 000 gene-enriched reads from maize were generated and clustered into Assembled Zea mays (AZM) sequences. Here we report the current AZM release, which consists of ∼298 Mb representing 243 807 sequence assemblies and singletons. In order to provide a repository of publicly available maize genomic sequences, we have created the TIGR Maize Database (). In this resource, we have assembled and annotated the AZMs and used available sequenced markers to anchor AZMs to maize chromosomes. We have constructed a maize repeat database and generated draft sequence assemblies of 287 maize bacterial artificial chromosome (BAC) clone sequences, which we annotated along with 172 additional publicly available BAC clones. All sequences, assemblies and annotations are available at the project website via web interfaces and FTP downloads