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Determining the genomic sequences of microorganisms is the basis and prerequisite for understanding their biology and functional characterization. While the advent of low-cost, extremely high-throughput second-generation sequencing technologies and the parallel development of assembly algorithms have generated rapid and cost-effective genome assemblies, such assemblies are often unfinished, fragmented draft genomes as a result of short read lengths and long repeats present in multiple copies. Third-generation, PacBio sequencing technologies circumvented this problem by greatly increasing read length. Hybrid approaches including ALLPATHS-LG, PacBio corrected reads pipeline, SPAdes, and SSPACE-LongRead, and non-hybrid approaches—hierarchical genome-assembly process (HGAP) and PacBio corrected reads pipeline via self-correction—have therefore been proposed to utilize the PacBio long reads that can span many thousands of bases to facilitate the assembly of complete microbial genomes. However, standardized procedures that aim at evaluating and comparing these approaches are currently insufficient. To address the issue, we herein provide a comprehensive comparison by collecting datasets for the comparative assessment on the above-mentioned five assemblers. In addition to offering explicit and beneficial recommendations to practitioners, this study aims to aid in the design of a paradigm positioned to complete bacterial genome assembly.

Summary. Two envelope glycoprotein (E rns and E2) regions of the classical swine fever virus (CSFV) were amplified by RT-PCR and sequenced directly from 158 specimens collected between 1989 and 2003 in Taiwan. Phylogenetic analysis of the two regions revealed a similar tree topology and the E rns region provided better discrimination than the E2 region. One hundred and fifteen isolates out of the 158 isolates were clustered within subgroup 2.1 (further classified as 2.1a and 2.1b) and 2.2, which were considered to be likely of the introduced strains, whereas the remaining 43 isolates were clustered within subgroup 3.4 and were considered to be of the endemic strains. The subgroup 2.1a viruses were first detected in 1994 and predominated from 1995 onwards. However, subgroup 3.4 viruses were prevalent in the early years, not being isolated after 1996. We have observed a dramatic switch in genotype from subgroup 3.4 to 2.1a. The subgroup 2.1a isolates are closely related to the Paderborn and Lao isolates, whereas 2.1b isolates have a close relationship to the Chinese Guangxi isolates. The phylogenetic tree of 27 CSFV sequences based on the complete envelope glycoprotein gene (E rns -E2) displayed better resolution than that based on the complete open reading frame.

Alkaline phosphatases (APs) catalyze the hydrolysis and transphosphorylation of phosphate monoesters. Quantum mechanical, molecular dynamics, and molecular docking techniques were applied to computationally model the catalytic mechanism of human placental AP (PLAP). Kinetic and thermodynamic evaluations were performed for each reaction step. The functional significances of the more important residues within the active site were analyzed. The role of the metal ion at the metal binding site M3 was also examined. The calculated activation and reaction energy and free energy values obtained suggested the nucleophilic attack of the Ser92 alkoxide on the phosphorus atom of the substrate would be the rate-limiting step of the catalytic hydrolysis of alkyl phosphate monoesters by PLAP. The reactivities of the wild-type M3-Mg enzyme and the M3-Zn protein were compared, and the main difference observed was a change in the coordination number of the M3 metal for the M3-Zn enzyme. This modification in the active site structure lowered the free energy profile for the second chemical step of the catalytic mechanism (hydrolysis of the covalent phosphoserine intermediate). Consequently, a greater stabilization of the phosphoseryl moiety resulted in a small increment in the activation free energy of the phosphoserine hydrolysis reaction. These computational results suggest that the activation of APs by magnesium at the M3 site is caused by the preference of Mg(2+) for octahedral coordination, which structurally stabilizes the active site into a catalytically most active conformation. The present theoretical results are in good agreement with previously reported experimental studies.