Library preparation methodology can influence genomic and functional predictions in human microbiome research.

Observations from human microbiome studies are often conflicting or inconclusive. Many factors likely contribute to these issues including small cohort sizes, sample collection, and handling and processing differences. The field of microbiome research is moving from 16S rDNA gene sequencing to a more comprehensive genomic and functional representation through whole-genome sequencing (WGS) of complete communities. Here we performed quantitative and qualitative analyses comparing WGS metagenomic data from human stool specimens using the Illumina Nextera XT and Illumina TruSeq DNA PCR-free kits, and the KAPA Biosystems Hyper Prep PCR and PCR-free systems. Significant differences in taxonomy are observed among the four different next-generation sequencing library preparations using a DNA mock community and a cell control of known concentration. We also revealed biases in error profiles, duplication rates, and loss of reads representing organisms that have a high %G+C content that can significantly impact results. As with all methods, the use of benchmarking controls has revealed critical differences among methods that impact sequencing results and later would impact study interpretation. We recommend that the community adopt PCR-free-based approaches to reduce PCR bias that affects calculations of abundance and to improve assemblies for accurate taxonomic assignment. Furthermore, the inclusion of a known-input cell spike-in control provides accurate quantitation of organisms in clinical samples

Replicating phages in the epidermal mucosa of the eel (anguilla anguilla)

n this work, we used the eel (Anguilla anguilla) as an animal model to test the hypothesis of Barr et al. (2013a,b) about the putative role of the epidermal mucosa as a phage enrichment layer. To this end, we analyzed the microbial content of the skin mucus of wild and farmed eels by using a metagenomic approach. We found a great abundance of replicating phage genomes (concatemers) in all the samples. They were assembled in four complete genomes of three Myovirus and one Podovirus. We also found evidences that ΦKZ and Podovirus phages could be part of the resident microbiota associated to the eel mucosal surface and persist on them over the time. Moreover, the viral abundance estimated by epiflorescent counts and by metagenomic recruitment from eel mucosa was higher than that of the surrounding water. Taken together, our results support the hypothesis that claims a possible role of phages in the animal mucus as agents controlling bacterial populations, including pathogenic species, providing a kind of innate immunity

FastGroup: A program to dereplicate libraries of 16S rDNA sequences

<p>Abstract</p> <p>Background</p> <p>Ribosomal 16S DNA sequences are an essential tool for identifying and classifying microbes. High-throughput DNA sequencing now makes it economically possible to produce very large datasets of 16S rDNA sequences in short time periods, necessitating new computer tools for analyses. Here we describe FastGroup, a Java program designed to dereplicate libraries of 16S rDNA sequences. By dereplication we mean to: 1) compare all the sequences in a data set to each other, 2) group similar sequences together, and 3) output a representative sequence from each group. In this way, duplicate sequences are removed from a library.</p> <p>Results</p> <p>FastGroup was tested using a library of single-pass, bacterial 16S rDNA sequences cloned from coral-associated bacteria. We found that the optimal strategy for dereplicating these sequences was to: 1) trim ambiguous bases from the 5' end of the sequences and all sequence 3' of the conserved Bact517 site, 2) match the sequences from the 3' end, and 3) group sequences >=97% identical to each other.</p> <p>Conclusions</p> <p>The FastGroup program simplifies the dereplication of 16S rDNA sequence libraries and prepares the raw sequences for subsequent analyses.</p

Hawai‘i-grown tea: a market feasibility study executive summary

This report is a feasability study for developing tea into a viable and sustainable industry for the state of Hawai‘i. It examines the economic potential of a Hawai‘i tea industry and provides guidelines for the development of the industry to a mature stage in the product's life cycle

Utilization Of Defined Microbial Communities Enables Effective Evaluation Of Meta-Genomic Assemblies

Background: Metagenomics is the study of the microbial genomes isolated from communities found on our bodies or in our environment. By correctly determining the relation between human health and the human associated microbial communities, novel mechanisms of health and disease can be found, thus enabling the development of novel diagnostics and therapeutics. Due to the diversity of the microbial communities, strategies developed for aligning human genomes cannot be utilized, and genomes of the microbial species in the community must be assembled de novo. However, in order to obtain the best metagenomic assemblies, it is important to choose the proper assembler. Due to the rapidly evolving nature of metagenomics, new assemblers are constantly created, and the field has not yet agreed on a standardized process. Furthermore, the truth sets used to compare these methods are either too simple (computationally derived diverse communities) or complex (microbial communities of unknown composition), yielding results that are hard to interpret. In this analysis, we interrogate the strengths and weaknesses of five popular assemblers through the use of defined biological samples of known genomic composition and abundance. We assessed the performance of each assembler on their ability to reassemble genomes, call taxonomic abundances, and recreate open reading frames (ORFs). Results: We tested five metagenomic assemblers: Omega, metaSPAdes, IDBA-UD, metaVelvet and MEGAHIT on known and synthetic metagenomic data sets. MetaSPAdes excelled in diverse sets, IDBA-UD performed well all around, metaVelvet had high accuracy in high abundance organisms, and MEGAHIT was able to accurately differentiate similar organisms within a community. At the ORF level, metaSPAdes and MEGAHIT had the least number of missing ORFs within diverse and similar communities respectively. Conclusions: Depending on the metagenomics question asked, the correct assembler for the task at hand will differ. It is important to choose the appropriate assembler, and thus clearly define the biological problemof an experiment, as different assemblers will give different answers to the same question

Genome Sequences of Two Closely Related Vibrio parahaemolyticus Phages, VP16T and VP16C

Two bacteriophages of an environmental isolate of Vibrio parahaemolyticus were isolated and sequenced. The VP16T and VP16C phages were separated from a mixed lysate based on plaque morphology and exhibit 73 to 88% sequence identity over about 80% of their genomes. Only about 25% of their predicted open reading frames are similar to genes with known functions in the GenBank database. Both phages have cos sites and open reading frames encoding proteins closely related to coliphage lambda's terminase protein (the large subunit). Like in coliphage lambda and other siphophages, a large operon in each phage appears to encode proteins involved in DNA packaging and capsid assembly and presumably in host lysis; we refer to this as the structural operon. In addition, both phages have open reading frames closely related to genes encoding DNA polymerase and helicase proteins. Both phages also encode several putative transcription regulators, an apparent polypeptide deformylase, and a protein related to a virulence-associated protein, VapE, of Dichelobacter nodosus. Despite the similarity of the proteins and genome organization, each of the phages also encodes a few proteins not encoded by the other. We did not identify genes closely related to genes encoding integrase proteins belonging to either the tyrosine or serine recombinase family, and we have no evidence so far that these phages can lysogenize the V. parahaemolyticus strain 16 host. Surprisingly for active lytic viruses, the two phages have a codon usage that is very different than that of the host, suggesting the possibility that they may be relative newcomers to growth in V. parahaemolyticus. The DNA sequences should allow us to characterize the lifestyles of VP16T and VP16C and the interactions between these phages and their host at the molecular level, as well as their relationships to other marine and nonmarine phages

Utilization of defined microbial communities enables effective evaluation of meta-genomic assemblies

Abstract Background Metagenomics is the study of the microbial genomes isolated from communities found on our bodies or in our environment. By correctly determining the relation between human health and the human associated microbial communities, novel mechanisms of health and disease can be found, thus enabling the development of novel diagnostics and therapeutics. Due to the diversity of the microbial communities, strategies developed for aligning human genomes cannot be utilized, and genomes of the microbial species in the community must be assembled de novo. However, in order to obtain the best metagenomic assemblies, it is important to choose the proper assembler. Due to the rapidly evolving nature of metagenomics, new assemblers are constantly created, and the field has not yet agreed on a standardized process. Furthermore, the truth sets used to compare these methods are either too simple (computationally derived diverse communities) or complex (microbial communities of unknown composition), yielding results that are hard to interpret. In this analysis, we interrogate the strengths and weaknesses of five popular assemblers through the use of defined biological samples of known genomic composition and abundance. We assessed the performance of each assembler on their ability to reassemble genomes, call taxonomic abundances, and recreate open reading frames (ORFs). Results We tested five metagenomic assemblers: Omega, metaSPAdes, IDBA-UD, metaVelvet and MEGAHIT on known and synthetic metagenomic data sets. MetaSPAdes excelled in diverse sets, IDBA-UD performed well all around, metaVelvet had high accuracy in high abundance organisms, and MEGAHIT was able to accurately differentiate similar organisms within a community. At the ORF level, metaSPAdes and MEGAHIT had the least number of missing ORFs within diverse and similar communities respectively. Conclusions Depending on the metagenomics question asked, the correct assembler for the task at hand will differ. It is important to choose the appropriate assembler, and thus clearly define the biological problem of an experiment, as different assemblers will give different answers to the same question