Complete Genome Sequence of Escherichia coli AS19, an Antibiotic-Sensitive Variant of E. coli Strain B REL606.

The chemically mutagenized Escherichia coli strain AS19 was isolated on the basis of its enhanced sensitivity to different antibiotics, in particular to actinomycin. The strain was later modified to study rRNA modifications that confer antibiotic resistance. Here, we present the genome sequence of the variant E. coli AS19-RrmA.

NCBI-compliant genome submissions: tips and tricks to save time and money.

Genome sequences nowadays play a central role in molecular biology and bioinformatics. These sequences are shared with the scientific community through sequence databases. The sequence repositories of the International Nucleotide Sequence Database Collaboration (INSDC, comprising GenBank, ENA and DDBJ) are the largest in the world. Preparing an annotated sequence in such a way that it will be accepted by the database is challenging because many validation criteria apply. In our opinion, it is an undesirable situation that researchers who want to submit their sequence need either a lot of experience or help from partners to get the job done. To save valuable time and money, we list a number of recommendations for people who want to submit an annotated genome to a sequence database, as well as for tool developers, who could help to ease the process.

SSPACE-LongRead: scaffolding bacterial draft genomes using long read sequence information.

BACKGROUND: The recent introduction of the Pacific Biosciences RS single molecule sequencing technology has opened new doors to scaffolding genome assemblies in a cost-effective manner. The long read sequence information is promised to enhance the quality of incomplete and inaccurate draft assemblies constructed from Next Generation Sequencing (NGS) data. RESULTS: Here we propose a novel hybrid assembly methodology that aims to scaffold pre-assembled contigs in an iterative manner using PacBio RS long read information as a backbone. On a test set comprising six bacterial draft genomes, assembled using either a single Illumina MiSeq or Roche 454 library, we show that even a 50× coverage of uncorrected PacBio RS long reads is sufficient to drastically reduce the number of contigs. Comparisons to the AHA scaffolder indicate our strategy is better capable of producing (nearly) complete bacterial genomes. CONCLUSIONS: The current work describes our SSPACE-LongRead software which is designed to upgrade incomplete draft genomes using single molecule sequences. We conclude that the recent advances of the PacBio sequencing technology and chemistry, in combination with the limited computational resources required to run our program, allow to scaffold genomes in a fast and reliable manner.

Bioinformatics approaches for viral metagenomics in plants using short RNAs: model case of study and application to a Cicer arietinum population.

Over the past years deep sequencing experiments have opened novel doors to reconstruct viral populations in a high-throughput and cost-effective manner. Currently a substantial number of studies have been performed which employ next generation sequencing techniques to either analyze known viruses by means of a reference-guided approach or to discover novel viruses using a de novo-based strategy. Taking advantage of the well-known Cymbidium ringspot virus we have carried out a comparison of different bioinformatics tools to reconstruct the viral genome based on 21-27 nt short (s)RNA sequencing with the aim to identify the most efficient pipeline. The same approach was applied to a population of plants constituting an ancient variety of Cicer arietinum with red seeds. Among the discovered viruses, we describe the presence of a Tobamovirus referring to the Tomato mottle mosaic virus (NC_022230), which was not yet observed on C. arietinum nor revealed in Europe and a viroid referring to Hop stunt viroid (NC_001351.1) never reported in chickpea. Notably, a reference sequence guided approach appeared the most efficient in such kind of investigation. Instead, the de novo assembly reached a non-appreciable coverage although the most prominent viral species could still be identified. Advantages and limitations of viral metagenomics analysis using sRNAs are discussed.

The king cobra genome reveals dynamic gene evolution and adaptation in the snake venom system.

Snakes are limbless predators, and many species use venom to help overpower relatively large, agile prey. Snake venoms are complex protein mixtures encoded by several multilocus gene families that function synergistically to cause incapacitation. To examine venom evolution, we sequenced and interrogated the genome of a venomous snake, the king cobra (Ophiophagus hannah), and compared it, together with our unique transcriptome, microRNA, and proteome datasets from this species, with data from other vertebrates. In contrast to the platypus, the only other venomous vertebrate with a sequenced genome, we find that snake toxin genes evolve through several distinct co-option mechanisms and exhibit surprisingly variable levels of gene duplication and directional selection that correlate with their functional importance in prey capture. The enigmatic accessory venom gland shows a very different pattern of toxin gene expression from the main venom gland and seems to have recruited toxin-like lectin genes repeatedly for new nontoxic functions. In addition, tissue-specific microRNA analyses suggested the co-option of core genetic regulatory components of the venom secretory system from a pancreatic origin. Although the king cobra is limbless, we recovered coding sequences for all Hox genes involved in amniote limb development, with the exception of Hoxd12. Our results provide a unique view of the origin and evolution of snake venom and reveal multiple genome-level adaptive responses to natural selection in this complex biological weapon system. More generally, they provide insight into mechanisms of protein evolution under strong selection.

First draft genome sequence of the Japanese eel, Anguilla japonica.

The Japanese eel is a much appreciated research object and very important for Asian aquaculture; however, its genomic resources are still limited. We have used a streamlined bioinformatics pipeline for the de novo assembly of the genome sequence of the Japanese eel from raw Illumina sequence reads. The total assembled genome has a size of 1.15 Gbp, which is divided over 323,776 scaffolds with an N50 of 52,849 bp, a minimum scaffold size of 200 bp and a maximum scaffold size of 1.14 Mbp. Direct comparison of a representative set of scaffolds revealed that all the Hox genes and their intergenic distances are almost perfectly conserved between the European and the Japanese eel. The first draft genome sequence of an organism strongly catalyzes research progress in multiple fields. Therefore, the Japanese eel genome sequence will provide a rich resource of data for all scientists working on this important fish species.

Toward almost closed genomes with GapFiller.

De novo assembly is a commonly used application of next-generation sequencing experiments. The ultimate goal is to puzzle millions of reads into one complete genome, although draft assemblies usually result in a number of gapped scaffold sequences. In this paper we propose an automated strategy, called GapFiller, to reliably close gaps within scaffolds using paired reads. The method shows good results on both bacterial and eukaryotic datasets, allowing only few errors. As a consequence, the amount of additional wetlab work needed to close a genome is drastically reduced. The software is available at http://www.baseclear.com/bioinformatics-tools/.

Scaffolding pre-assembled contigs using SSPACE.

SUMMARY: De novo assembly tools play a main role in reconstructing genomes from next-generation sequencing (NGS) data and usually yield a number of contigs. Using paired-read sequencing data it is possible to assess the order, distance and orientation of contigs and combine them into so-called scaffolds. Although the latter process is a crucial step in finishing genomes, scaffolding algorithms are often built-in functions in de novo assembly tools and cannot be independently controlled. We here present a new tool, called SSPACE, which is a stand-alone scaffolder of pre-assembled contigs using paired-read data. Main features are: a short runtime, multiple library input of paired-end and/or mate pair datasets and possible contig extension with unmapped sequence reads. SSPACE shows promising results on both prokaryote and eukaryote genomic testsets where the amount of initial contigs was reduced by at least 75%.

Integrated analysis of DNA copy number and gene expression microarray data using gene sets.

BACKGROUND: Genes that play an important role in tumorigenesis are expected to show association between DNA copy number and RNA expression. Optimal power to find such associations can only be achieved if analysing copy number and gene expression jointly. Furthermore, some copy number changes extend over larger chromosomal regions affecting the expression levels of multiple resident genes. RESULTS: We propose to analyse copy number and expression array data using gene sets, rather than individual genes. The proposed model is robust and sensitive. We re-analysed two publicly available datasets as illustration. These two independent breast cancer datasets yielded similar patterns of association between gene dosage and gene expression levels, in spite of different platforms having been used. Our comparisons show a clear advantage to using sets of genes' expressions to detect associations with long-spanning, low-amplitude copy number aberrations. In addition, our model allows for using additional explanatory variables and does not require mapping between copy number and expression probes. CONCLUSION: We developed a general and flexible tool for integration of multiple microarray data sets, and showed how the identification of genes whose expression is affected by copy number aberrations provides a powerful approach to prioritize putative targets for functional validation.