July 19, 2019  |  

Mind the gap: upgrading genomes with Pacific Biosciences RS long-read sequencing technology.

Many genomes have been sequenced to high-quality draft status using Sanger capillary electrophoresis and/or newer short-read sequence data and whole genome assembly techniques. However, even the best draft genomes contain gaps and other imperfections due to limitations in the input data and the techniques used to build draft assemblies. Sequencing biases, repetitive genomic features, genomic polymorphism, and other complicating factors all come together to make some regions difficult or impossible to assemble. Traditionally, draft genomes were upgraded to “phase 3 finished” status using time-consuming and expensive Sanger-based manual finishing processes. For more facile assembly and automated finishing of draft genomes, we present here an automated approach to finishing using long-reads from the Pacific Biosciences RS (PacBio) platform. Our algorithm and associated software tool, PBJelly, (publicly available at https://sourceforge.net/projects/pb-jelly/) automates the finishing process using long sequence reads in a reference-guided assembly process. PBJelly also provides “lift-over” co-ordinate tables to easily port existing annotations to the upgraded assembly. Using PBJelly and long PacBio reads, we upgraded the draft genome sequences of a simulated Drosophila melanogaster, the version 2 draft Drosophila pseudoobscura, an assembly of the Assemblathon 2.0 budgerigar dataset, and a preliminary assembly of the Sooty mangabey. With 24× mapped coverage of PacBio long-reads, we addressed 99% of gaps and were able to close 69% and improve 12% of all gaps in D. pseudoobscura. With 4× mapped coverage of PacBio long-reads we saw reads address 63% of gaps in our budgerigar assembly, of which 32% were closed and 63% improved. With 6.8× mapped coverage of mangabey PacBio long-reads we addressed 97% of gaps and closed 66% of addressed gaps and improved 19%. The accuracy of gap closure was validated by comparison to Sanger sequencing on gaps from the original D. pseudoobscura draft assembly and shown to be dependent on initial reference quality.


July 7, 2019  |  

Comparative genome analysis of Pseudomonas knackmussii B13, the first bacterium known to degrade chloroaromatic compounds.

Pseudomonas knackmussii B13 was the first strain to be isolated in 1974 that could degrade chlorinated aromatic hydrocarbons. This discovery was the prologue for subsequent characterization of numerous bacterial metabolic pathways, for genetic and biochemical studies, and which spurred ideas for pollutant bioremediation. In this study, we determined the complete genome sequence of B13 using next generation sequencing technologies and optical mapping. Genome annotation indicated that B13 has a variety of metabolic pathways for degrading monoaromatic hydrocarbons including chlorobenzoate, aminophenol, anthranilate and hydroxyquinol, but not polyaromatic compounds. Comparative genome analysis revealed that B13 is closest to Pseudomonas denitrificans and Pseudomonas aeruginosa. The B13 genome contains at least eight genomic islands [prophages and integrative conjugative elements (ICEs)], which were absent in closely related pseudomonads. We confirm that two ICEs are identical copies of the 103?kb self-transmissible element ICEclc that carries the genes for chlorocatechol metabolism. Comparison of ICEclc showed that it is composed of a variable and a ‘core’ region, which is very conserved among proteobacterial genomes, suggesting a widely distributed family of so far uncharacterized ICE. Resequencing of two spontaneous B13 mutants revealed a number of single nucleotide substitutions, as well as excision of a large 220?kb region and a prophage that drastically change the host metabolic capacity and survivability. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.


July 7, 2019  |  

Complete genome sequence of the chromate-reducing bacterium Thermoanaerobacter thermohydrosulfuricus strain BSB-33.

Thermoanaerobacter thermohydrosulfuricus BSB-33 is a thermophilic gram positive obligate anaerobe isolated from a hot spring in West Bengal, India. Unlike other T. thermohydrosulfuricus strains, BSB-33 is able to anaerobically reduce Fe(III) and Cr(VI) optimally at 60 °C. BSB-33 is the first Cr(VI) reducing T. thermohydrosulfuricus genome sequenced and of particular interest for bioremediation of environmental chromium contaminations. Here we discuss features of T. thermohydrosulfuricus BSB-33 and the unique genetic elements that may account for the peculiar metal reducing properties of this organism. The T. thermohydrosulfuricus BSB-33 genome comprises 2597606 bp encoding 2581 protein genes, 12 rRNA, 193 pseudogenes and has a G?+?C content of 34.20 %. Putative chromate reductases were identified by comparative analyses with other Thermoanaerobacter and chromate-reducing bacteria.


July 7, 2019  |  

High-quality permanent draft genome sequence of the Lebeckia ambigua-nodulating Burkholderia sp. strain WSM4176.

Burkholderia sp. strain WSM4176 is an aerobic, motile, Gram-negative, non-spore-forming rod that was isolated from an effective N2-fixing root nodule of Lebeckia ambigua collected in Nieuwoudtville, Western Cape of South Africa, in October 2007. This plant persists in infertile, acidic and deep sandy soils, and is therefore an ideal candidate for a perennial based agriculture system in Western Australia. Here we describe the features of Burkholderia sp. strain WSM4176, which represents a potential inoculant quality strain for L. ambigua, together with sequence and annotation. The 9,065,247 bp high-quality-draft genome is arranged in 13 scaffolds of 65 contigs, contains 8369 protein-coding genes and 128 RNA-only encoding genes, and is part of the GEBA-RNB project proposal (Project ID 882).


July 7, 2019  |  

Stenotrophomonas comparative genomics reveals genes and functions that differentiate beneficial and pathogenic bacteria.

In recent years, the number of human infections caused by opportunistic pathogens has increased dramatically. Plant rhizospheres are one of the most typical natural reservoirs for these pathogens but they also represent a great source for beneficial microbes with potential for biotechnological applications. However, understanding the natural variation and possible differences between pathogens and beneficials is the main challenge in furthering these possibilities. The genus Stenotrophomonas contains representatives found to be associated with human and plant host.We used comparative genomics as well as transcriptomic and physiological approaches to detect significant borders between the Stenotrophomonas strains: the multi-drug resistant pathogenic S. maltophilia and the plant-associated strains S. maltophilia R551-3 and S. rhizophila DSM14405T (both are biocontrol agents). We found an overall high degree of sequence similarity between the genomes of all three strains. Despite the notable similarity in potential factors responsible for host invasion and antibiotic resistance, other factors including several crucial virulence factors and heat shock proteins were absent in the plant-associated DSM14405T. Instead, S. rhizophila DSM14405T possessed unique genes for the synthesis and transport of the plant-protective spermidine, plant cell-wall degrading enzymes, and high salinity tolerance. Moreover, the presence or absence of bacterial growth at 37°C was identified as a very simple method in differentiating between pathogenic and non-pathogenic isolates. DSM14405T is not able to grow at this human-relevant temperature, most likely in great part due to the absence of heat shock genes and perhaps also because of the up-regulation at increased temperatures of several genes involved in a suicide mechanism.While this study is important for understanding the mechanisms behind the emerging pattern of infectious diseases, it is, to our knowledge, the first of its kind to assess the risk of beneficial strains for biotechnological applications. We identified certain traits typical of pathogens such as growth at the human body temperature together with the production of heat shock proteins as opposed to a temperature-regulated suicide system that is harnessed by beneficials.


July 7, 2019  |  

FGAP: an automated gap closing tool.

The fast reduction of prices of DNA sequencing allowed rapid accumulation of genome data. However, the process of obtaining complete genome sequences is still very time consuming and labor demanding. In addition, data produced from various sequencing technologies or alternative assemblies remain underexplored to improve assembly of incomplete genome sequences.We have developed FGAP, a tool for closing gaps of draft genome sequences that takes advantage of different datasets. FGAP uses BLAST to align multiple contigs against a draft genome assembly aiming to find sequences that overlap gaps. The algorithm selects the best sequence to fill and eliminate the gap.FGAP reduced the number of gaps by 78% in an E. coli draft genome assembly using two different sequencing technologies, Illumina and 454. Using PacBio long reads, 98% of gaps were solved. In human chromosome 14 assemblies, FGAP reduced the number of gaps by 35%. All the inserted sequences were validated with a reference genome using QUAST. The source code and a web tool are available at http://www.bioinfo.ufpr.br/fgap/.


July 7, 2019  |  

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

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.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.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.


July 7, 2019  |  

Permanent draft genome sequences of the symbiotic nitrogen fixing Ensifer meliloti strains BO21CC and AK58.

Ensifer (syn. Sinorhizobium) meliloti is an important symbiotic bacterial species that fixes nitrogen. Strains BO21CC and AK58 were previously investigated for their substrate utilization and their plant-growth promoting abilities showing interesting features. Here, we describe the complete genome sequence and annotation of these strains. BO21CC and AK58 genomes are 6,985,065 and 6,974,333 bp long with 6,746 and 6,992 genes predicted, respectively.


July 7, 2019  |  

Improving genome assemblies by sequencing PCR products with PacBio.

Advances in sequencing technologies have dramatically reduced costs in producing high-quality draft genomes. However, there are still many contigs and possible misassembled regions in those draft genomes. Improving the quality of these genomes requires an efficient and economical means to close gaps and resequence some regions. Sequencing pooled gap region PCR products with Pacific Biosciences (PacBio) provides a significantly less expensive means for this need. We have developed a genome improvement pipeline with this strategy after decreasing a loading bias against larger PCR products in the PacBio process. Compared with Sanger technology, this approach is not only cost-effective but also can close gaps greater than 2.5 kb in a single round of reactions, and sequence through high GC regions as well as difficult secondary structures such as small hairpin loops.


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