Efficient de novo assembly of large and complex genomes by massively parallel sequencing of Fosmid pools

Background

Sampling genomes with Fosmid vectors and sequencing of pooled Fosmid libraries on the Illumina platform for massive parallel sequencing is a novel and promising approach to optimizing the trade-off between sequencing costs and assembly quality.

Results

In order to sequence the genome of Norway spruce, which is of great size and complexity, we developed and applied a new technology based on the massive production, sequencing, and assembly of Fosmid pools (FP). The spruce chromosomes were sampled with ~40,000 bp Fosmid inserts to obtain around two-fold genome coverage, in parallel with traditional whole genome shotgun sequencing (WGS) of haploid and diploid genomes. Compared to the WGS results, the contiguity and quality of the FP assemblies were high, and they allowed us to fill WGS gaps resulting from repeats, low coverage, and allelic differences. The FP contig sets were further merged with WGS data using a novel software package GAM-NGS.

Conclusions

By exploiting FP technology, the first published assembly of a conifer genome was sequenced entirely with massively parallel sequencing. Here we provide a comprehensive report on the different features of the approach and the optimization of the process.We have made public the input data (FASTQ format) for the set of pools used in this study:ftp://congenie.org/congenie/Nystedt_2013/Assembly/ProcessedData/FosmidPools/.(alternatively accessible via http://congenie.org/downloads).The software used for running the assembly process is available at http://research.scilifelab.se/andrej_alexeyenko/downloads/fpools/.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-439) contains supplementary material, which is available to authorized users.     

Draft genome sequence of marine alphaproteobacterial strain HIMB11, the first cultivated representative of a unique lineage within the Roseobacter clade possessing an unusually small genome

Strain HIMB11 is a planktonic marine bacterium isolated from coastal seawater in Kaneohe Bay, Oahu, Hawaii belonging to the ubiquitous and versatile Roseobacter clade of the alphaproteobacterial family Rhodobacteraceae. Here we describe the preliminary characteristics of strain HIMB11, including annotation of the draft genome sequence and comparative genomic analysis with other members of the Roseobacter lineage. The 3,098,747 bp draft genome is arranged in 34 contigs and contains 3,183 protein-coding genes and 54 RNA genes. Phylogenomic and 16S rRNA gene analyses indicate that HIMB11 represents a unique sublineage within the Roseobacter clade. Comparison with other publicly available genome sequences from members of the Roseobacter lineage reveals that strain HIMB11 has the genomic potential to utilize a wide variety of energy sources (e.g. organic matter, reduced inorganic sulfur, light, carbon monoxide), while possessing a reduced number of substrate transporters.     

Genome sequence of the chemoheterotrophic soil bacterium Saccharomonospora cyanea type strain (NA-134T)

Saccharomonospora cyanea Runmao et al. 1988 is a member of the genus Saccharomonospora in the family Pseudonocardiaceae that is moderately well characterized at the genome level thus far. Members of the genus Saccharomonospora are of interest because they originate from diverse habitats, such as soil, leaf litter, manure, compost, surface of peat, moist, over-heated grain, and ocean sediment, where they probably play a role in the primary degradation of plant material by attacking hemicellulose. Species of the genus Saccharomonospora are usually Gram-positive, non-acid fast, and are classified among the actinomycetes. S. cyanea is characterized by a dark blue (= cyan blue) aerial mycelium. After S. viridis, S. azurea, and S. marina, S. cyanea is only the fourth member in the genus for which a completely sequenced (non-contiguous finished draft status) type strain genome will be published. Here we describe the features of this organism, together with the draft genome sequence, and annotation. The 5,408,301 bp long chromosome with its 5,139 protein-coding and 57 RNA genes was sequenced as part of the DOE funded Community Sequencing Program (CSP) 2010 at the Joint Genome Institute (JGI).     

Non-contiguous finished genome sequence and description of Fenollaria massiliensis gen. nov., sp. nov., a new genus of anaerobic bacterium

Fenollaria massiliensis strain 9401234^T, is the type strain of Fenollaria massiliensis gen. nov., sp. nov., a new species within a new genus Fenollaria. This strain, whose genome is described here, was isolated from an osteoarticular sample. F. massiliensis strain 9401234^T is an obligate anaerobic Gram-negative bacillus. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 1.71 Mbp long genome exhibits a G+C content of 34.46% and contains 1,667 protein-coding and 30 RNA genes, including 3 rRNA genes.     

Non-contiguous finished genome sequence and description of Oceanobacillus massiliensis sp. nov.

Oceanobacillus massiliensis strain N’Diop^T sp. nov. is the type strain of O. massiliensis sp. nov., a new species within the genus Oceanobacillus. This strain, whose genome is described here, was isolated from the fecal flora of a healthy patient. O. massiliensis is an aerobic rod. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 3,532,675 bp long genome contains 3,519 protein-coding genes and 72 RNA genes, including between 6 and 8 rRNA operons.     

Genome sequence of the chromate-resistant bacterium Leucobacter salsicius type strain M1-8T

Leucobacter salsicius M1-8^T is a member of the Microbacteriaceae family within the class Actinomycetales. This strain is a Gram-positive, rod-shaped bacterium and was previously isolated from a Korean fermented food. Most members of the genus Leucobacter are chromate-resistant and this feature could be exploited in biotechnological applications. However, the genus Leucobacter is poorly characterized at the genome level, despite its potential importance. Thus, the present study determined the features of Leucobacter salsicius M1-8^T, as well as its genome sequence and annotation. The genome comprised 3,185,418 bp with a G+C content of 64.5%, which included 2,865 protein-coding genes and 68 RNA genes. This strain possessed two predicted genes associated with chromate resistance, which might facilitate its growth in heavy metal-rich environments.     

Genome sequence of the clover-nodulating Rhizobium leguminosarum bv. trifolii strain SRDI943.

Rhizobium leguminosarum bv. trifolii SRDI943 (strain syn. V2-2) is an aerobic, motile, Gram-negative, non-spore-forming rod that was isolated from a root nodule of Trifolium michelianum Savi cv. Paradana that had been grown in soil collected from a mixed pasture in Victoria, Australia. This isolate was found to have a broad clover host range but was sub-optimal for nitrogen fixation with T. subterraneum (fixing 20-54% of reference inoculant strain WSM1325) and was found to be totally ineffective with the clover species T. polymorphum and T. pratense. Here we describe the features of R. leguminosarum bv. trifolii strain SRDI943, together with genome sequence information and annotation. The 7,412,387 bp high-quality-draft genome is arranged into 5 scaffolds of 5 contigs, contains 7,317 protein-coding genes and 89 RNA-only encoding genes, and is one of 100 rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project.     

Draft genome sequence of Rhodococcus rhodochrous strain ATCC 17895

Rhodococcus rhodochrous ATCC 17895 possesses an array of mono- and dioxygenases, as well as hydratases, which makes it an interesting organism for biocatalysis. R. rhodochrous is a Gram-positive aerobic bacterium with a rod-like morphology. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 6,869,887 bp long genome contains 6,609 protein-coding genes and 53 RNA genes. Based on small subunit rRNA analysis, the strain is more likely to be a strain of Rhodococcus erythropolis rather than Rhodococcus rhodochrous.     

Genome sequence of the free-living aerobic spirochete Turneriella parva type strain (HT), and emendation of the species Turneriella parva

Turneriella parva Levett et al. 2005 is the only species of the genus Turneriella which was established as a result of the reclassification of Leptospira parva Hovind-Hougen et al. 1982. Together with Leptonema and Leptospira, Turneriella constitutes the family Leptospiraceae, within the order Spirochaetales. Here we describe the features of this free-living aerobic spirochete together with the complete genome sequence and annotation. This is the first complete genome sequence of a member of the genus Turneriella and the 13^th member of the family Leptospiraceae for which a complete or draft genome sequence is now available. The 4,409,302 bp long genome with its 4,169 protein-coding and 45 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Phyto-mellitus: A phyto-chemical database for diabetes

Herbs are the base used for treatment in Ayurveda. We describe a database named Phyto-Mellitus with information on plants traditionally used for diabetes with their chemical constituents. The active principles of these plants are antioxidant and free radical scavenging.

Availability

http://www.bicmlacw.org/bt/     

Draft genome sequence of Dyadobacter tibetensis type strain (Y620-1) isolated from glacial ice

Dyadobacter tibetensis Y620-1 is the type strain of the species Dyadobacter tibetensis, isolated from ice at a depth of 59 m from a high altitude glacier in China (5670 m above sea level). It is psychrotolerant with growth temperature ranges of 4 to 35°C. Here we describe the features of this organism, together with the draft genome sequence and annotation. The 5,313,963 bp long genome contains 4,828 protein-coding genes and 39 RNA genes. To the best of our knowledge, this is the first Dyadobacter strain that was isolated from glacial ice. This study provides genetic information of this organism to identify the genes linked to its specific mechanisms for adaption to extreme glacial environment.     

Non-contiguous finished genome sequence and description of Brevibacillus massiliensis sp. nov.

Brevibacillus massiliensis strain phR^T sp. nov. is the type strain of B. massiliensis sp. nov., a new species within the genus Brevibacillus. This strain was isolated from the fecal flora of a woman suffering from morbid obesity. B. massiliensis is a Gram-positive aerobic rod-shaped bacterium. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 5,051,018 bp long genome (1 chromosome but no plasmid) contains 5,051 protein-coding and 84 RNA genes, and exhibits a G+C content of 53.1%.     

Complete genome sequence of Coriobacterium glomerans type strain (PW2T) from the midgut of Pyrrhocoris apterus L. (red soldier bug)

Coriobacterium glomerans Haas and König 1988, is the only species of the genus Coriobacterium, family Coriobacteriaceae, order Coriobacteriales, phylum Actinobacteria. The bacterium thrives as an endosymbiont of pyrrhocorid bugs, i.e. the red fire bug Pyrrhocoris apterus L. The rationale for sequencing the genome of strain PW2^T is its endosymbiotic life style which is rare among members of Actinobacteria. Here we describe the features of this symbiont, together with the complete genome sequence and its annotation. This is the first complete genome sequence of a member of the genus Coriobacterium and the sixth member of the order Coriobacteriales for which complete genome sequences are now available. The 2,115,681 bp long single replicon genome with its 1,804 protein-coding and 54 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Genome sequence of the Litoreibacter arenae type strain (DSM 19593T), a member of the Roseobacter clade isolated from sea sand

Litoreibacter arenae Kim et al. 2012 is a member of the genomically well-characterized Rhodobacteraceae clade within the Roseobacter clade. Representatives of this clade are known to be metabolically versatile and involved in marine carbon-producing and biogeochemical processes. They form a physiologically heterogeneous group of Alphaproteobacteria and were mostly found in coastal or polar waters, especially in symbiosis with algae, in microbial mats, in sediments or together with invertebrates and vertebrates. Here we describe the features of L. arenae DSM 19593^T, including novel aspects of its phenotype, together with the draft genome sequence and annotation. The 3,690,113 bp long genome consists of 17 scaffolds with 3,601 protein-coding and 56 RNA genes. This genome was sequenced as part of the activities of the Transregional Collaborative Research Centre 51 funded by the German Research Foundation (DFG).     

MendeLIMS: a web-based laboratory information management system for clinical genome sequencing

Background

Large clinical genomics studies using next generation DNA sequencing require the ability to select and track samples from a large population of patients through many experimental steps. With the number of clinical genome sequencing studies increasing, it is critical to maintain adequate laboratory information management systems to manage the thousands of patient samples that are subject to this type of genetic analysis.

Results

To meet the needs of clinical population studies using genome sequencing, we developed a web-based laboratory information management system (LIMS) with a flexible configuration that is adaptable to continuously evolving experimental protocols of next generation DNA sequencing technologies. Our system is referred to as MendeLIMS, is easily implemented with open source tools and is also highly configurable and extensible. MendeLIMS has been invaluable in the management of our clinical genome sequencing studies.

Conclusions

We maintain a publicly available demonstration version of the application for evaluation purposes at http://mendelims.stanford.edu. MendeLIMS is programmed in Ruby on Rails (RoR) and accesses data stored in SQL-compliant relational databases. Software is freely available for non-commercial use at http://dna-discovery.stanford.edu/software/mendelims/.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-290) contains supplementary material, which is available to authorized users.     

Non-contiguous genome sequence of Mycobacterium simiae strain DSM 44165T

Mycobacterium simiae is a non-tuberculosis mycobacterium causing pulmonary infections in both immunocompetent and imunocompromized patients. We announce the draft genome sequence of M. simiae DSM 44165^T. The 5,782,968-bp long genome with 65.15% GC content (one chromosome, no plasmid) contains 5,727 open reading frames (33% with unknown function and 11 ORFs sizing more than 5000 -bp), three rRNA operons, 52 tRNA, one 66-bp tmRNA matching with tmRNA tags from Mycobacterium avium, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium microti, Mycobacterium marinum, and Mycobacterium africanum and 389 DNA repetitive sequences. Comparing ORFs and size distribution between M. simiae and five other Mycobacterium species M. simiae clustered with M. abscessus and M. smegmatis. A 40-kb prophage was predicted in addition to two prophage-like elements, 7-kb and 18-kb in size, but no mycobacteriophage was seen after the observation of 10⁶ M. simiae cells. Fifteen putative CRISPRs were found. Three genes were predicted to encode resistance to aminoglycosides, betalactams and macrolide-lincosamide-streptogramin B. A total of 163 CAZYmes were annotated. M. simiae contains ESX-1 to ESX-5 genes encoding for a type-VII secretion system. Availability of the genome sequence may help depict the unique properties of this environmental, opportunistic pathogen.     

Non contiguous-finished genome sequence and description of Peptoniphilus senegalensis sp. nov.

Peptoniphilus senegalensis strain JC140^T sp. nov., is the type strain of P. senegalensis sp. nov., a new species within the genus Peptoniphilus. This strain, whose genome is described here, was isolated from the fecal flora of a healthy patient. P. senegalensis strain JC140^T is an obligate Gram-positive anaerobic coccus. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 1,840,641 bp long genome (1 chromosome but no plasmid) exhibits a G+C content of 32.2% and contains 1,744 protein-coding and 23 RNA genes, including 3 rRNA genes.Key words: Peptoniphilus senegalensis, genome     

Assembling allopolyploid genomes: no longer formidable

A combined approach of whole genome shotgun sequencing and ultra-high density linkage mapping using skim sequencing of a segregating population is effective for assembling allopolyploid genomes.See related Research, http://dx.doi.org/10.1186/s13059-015-0582-8     

Genome sequence of the phylogenetically isolated spirochete Leptonema illini type strain (3055T)

Leptonema illini Hovind-Hougen 1979 is the type species of the genus Leptonema, family Leptospiraceae, phylum Spirochaetes. Organisms of this family have a Gram-negative-like cell envelope consisting of a cytoplasmic membrane and an outer membrane. The peptidoglycan layer is associated with the cytoplasmic rather than the outer membrane. The two flagella of members of Leptospiraceae extend from the cytoplasmic membrane at the ends of the bacteria into the periplasmic space and are necessary for their motility. Here we describe the features of the L. illini type strain, together with the complete genome sequence, and annotation. This is the first genome sequence (finished at the level of Improved High Quality Draft) to be reported from of a member of the genus Leptonema and a representative of the third genus of the family Leptospiraceae for which complete or draft genome sequences are now available. The three scaffolds of the 4,522,760 bp draft genome sequence reported here, and its 4,230 protein-coding and 47 RNA genes are part of the Genomic Encyclopedia of Bacteria and Archaea project.