The recent advent of DNA sequencing technologies facilitates the use of genome sequencing data that provide means for more informative and precise classification and identification of members of the Bacteria and Archaea. Because the current species definition is based on the comparison of genome sequences between type and other strains in a given species, building a genome database with correct taxonomic information is of paramount need to enhance our efforts in exploring prokaryotic diversity and discovering novel species as well as for routine identifications. Here we introduce an integrated database, called EzBioCloud, that holds the taxonomic hierarchy of the Bacteria and Archaea, which is represented by quality-controlled 16S rRNA gene and genome sequences. Whole-genome assemblies in the NCBI Assembly Database were screened for low quality and subjected to a composite identification bioinformatics pipeline that employs gene-based searches followed by the calculation of average nucleotide identity. As a result, the database is made of 61 700 species/phylotypes, including 13 132 with validly published names, and 62 362 whole-genome assemblies that were identified taxonomically at the genus, species and subspecies levels. Genomic properties, such as genome size and DNA G+C content, and the occurrence in human microbiome data were calculated for each genus or higher taxa. This united database of taxonomy, 16S rRNA gene and genome sequences, with accompanying bioinformatics tools, should accelerate genome-based classification and identification of members of the Bacteria and Archaea. The database and related search tools are available at www.ezbiocloud.net/.
Average nucleotide identity (ANI) is a category of computational analysis that can be used to define species boundaries of Archaea and Bacteria. Calculating ANI usually involves the fragmentation of genome sequences, followed by nucleotide sequence search, alignment, and identity calculation. The original algorithm to calculate ANI used the BLAST program as its search engine. An improved ANI algorithm, called OrthoANI, was developed to accommodate the concept of orthology. Here, we compared four algorithms to compute ANI, namely ANIb (ANI algorithm using BLAST), ANIm (ANI using MUMmer), OrthoANIb (OrthoANI using BLAST) and OrthoANIu (OrthoANI using USEARCH) using >100,000 pairs of genomes with various genome sizes. By comparing values to the ANIb that is considered a standard, OrthoANIb and OrthoANIu exhibited good correlation in the whole range of ANI values. ANIm showed poor correlation for ANI of <90%. ANIm and OrthoANIu runs faster than ANIb by an order of magnitude. When genomes that are larger than 7 Mbp were analysed, the run-times of ANIm and OrthoANIu were shorter than that of ANIb by 53- and 22-fold, respectively. In conclusion, ANI calculation can be greatly sped up by the OrthoANIu method without losing accuracy. A web-service that can be used to calculate OrthoANIu between a pair of genome sequences is available at http://www.ezbiocloud.net/tools/ani . For large-scale calculation and integration in bioinformatics pipelines, a standalone JAVA program is available for download at http://www.ezbiocloud.net/tools/orthoaniu .
The cost of DNA sequencing has decreased due to advancements in Next Generation Sequencing. The number of sequences obtained from the Illumina platform is large, use of this platform can reduce costs more than the 454 pyrosequencer. However, the Illumina platform has other challenges, including bioinformatics analysis of large numbers of sequences and the need to reduce erroneous nucleotides generated at the 3'-ends of the sequences. These erroneous sequences can lead to errors in analysis of microbial communities. Therefore, correction of these erroneous sequences is necessary for accurate taxonomic identification. Several studies that have used the Illumina platform to perform metagenomic analyses proposed curating pipelines to increase accuracy. In this study, we evaluated the likelihood of obtaining an erroneous microbial composition using the MiSeq 250 bp paired sequence platform and improved the pipeline to reduce erroneous identifications. We compared different sequencing conditions by varying the percentage of control phiX added, the concentration of the sequencing library, and the 16S rRNA gene target region using a mock community sample composed of known sequences. Our recommended method corrected erroneous nucleotides and improved identification accuracy. Overall, 99.5% of the total reads shared 95% similarity with the corresponding template sequences and 93.6% of the total reads shared over 97% similarity. This indicated that the MiSeq platform can be used to analyze microbial communities at the genus level with high accuracy. The improved analysis method recommended in this study can be applied to amplicon studies in various environments using high-throughput reads generated on the MiSeq platform.
For a family-based sample, the phenotypic variance-covariance matrix can be parameterized to include the variance of a polygenic effect that has then been estimated using a variance component analysis. However, with the advent of large-scale genomic data, the genetic relationship matrix (GRM) can be estimated and can be utilized to parameterize the variance of a polygenic effect for population-based samples. Therefore narrow sense heritability, which is both population and trait specific, can be estimated with both population- and family-based samples. In this study we estimate heritability from both family-based and population-based samples, collected in Korea, and the heritability estimates from the pooled samples were, for height, 0.60; body mass index (BMI), 0.32; log-transformed triglycerides (log TG), 0.24; total cholesterol (TCHL), 0.30; high-density lipoprotein (HDL), 0.38; low-density lipoprotein (LDL), 0.29; systolic blood pressure (SBP), 0.23; and diastolic blood pressure (DBP), 0.24. Furthermore, we found differences in how heritability is estimated—in particular the amount of variance attributable to common environment in twins can be substantial—which indicates heritability estimates should be interpreted with caution.
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