Dataset Information

Analysis and comparison of the wolf microbiome under different environmental factors using three different data of Next Generation Sequencing.

ABSTRACT: Next Generation Sequencing has been widely used to characterize the prevalence of fecal bacteria in many different species. In this study, we attempted to employ a low-cost and high-throughput sequencing model to discern information pertaining to the wolf microbiota. It is hoped that this model will allow researchers to elucidate potential protective factors in relation to endangered wolf species. We propose three high-throughput sequencing models to reveal information pertaining to the micro-ecology of the wolf. Our analyses advised that, among the three models, more than 100,000 sequences are more appropriate to retrieve the communities' richness and diversity of micro-ecology. In addition, the top five wolf microbiome OTUs (99%) were members of the following five phyla: Bacteroidetes, Fusobacteria, Firmicutes, Proteobacteria, and Actinobacteria. While Alloprevotella, Clostridium_sensu_stricto_1, Anaerobiospirillum, Faecalibactreium and Streptococcus were shared by all samples, their relative abundances were differentially represented between domestic dogs and other wolves. Our findings suggest that altitude, human interference, age, and climate all contribute towards the micro-ecology of the wolf. Specifically, we observed that genera Succinivibrio and Turicibacter are significantly related to altitude and human interference (including hunting practices).

SUBMITTER: Wu X

PROVIDER: S-EPMC5596057 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Analysis and comparison of the wolf microbiome under different environmental factors using three different data of Next Generation Sequencing.

Wu Xiaoyang X Zhang Huanxin H Chen Jun J Shang Shuai S Yan Jiakuo J Chen Yao Y Tang Xuexi X Zhang Honghai H

Scientific reports 20170912 1

Next Generation Sequencing has been widely used to characterize the prevalence of fecal bacteria in many different species. In this study, we attempted to employ a low-cost and high-throughput sequencing model to discern information pertaining to the wolf microbiota. It is hoped that this model will allow researchers to elucidate potential protective factors in relation to endangered wolf species. We propose three high-throughput sequencing models to reveal information pertaining to the micro-ec ...[more]

PMID: 28900198

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Project description:BACKGROUND: The introduction of benchtop sequencers has made adoption of whole genome sequencing possible for a broader community of researchers than ever before. Concurrently, metagenomic sequencing (MGS) is rapidly emerging as a tool for interrogating complex samples that defy conventional analyses. In addition, next-generation sequencers are increasingly being used in clinical or related settings, for instance to track outbreaks. However, information regarding the analytical sensitivity or limit of detection (LoD) of benchtop sequencers is currently lacking. Furthermore, the specificity of sequence information at or near the LoD is unknown. RESULTS: In the present study, we assess the ability of three next-generation sequencing platforms to identify a pathogen (viral or bacterial) present in low titers in a clinically relevant sample (blood). Our results indicate that the Roche-454 Titanium platform is capable of detecting Dengue virus at titers as low as 1X102.5 pfu/mL, corresponding to an estimated 5.4X104 genome copies/ml maximum. The increased throughput of the benchtop sequencers, the Ion Torrent PGM and Illumina MiSeq platforms, enabled detection of viral genomes at concentrations as low as 1X104 genome copies/mL. Platform-specific biases were evident in sequence read distributions as well as viral genome coverage. For bacterial samples, only the MiSeq platform was able to provide sequencing reads that could be unambiguously classified as originating from Bacillus anthracis. CONCLUSION: The analytical sensitivity of all three platforms approaches that of standard qPCR assays. Although all platforms were able to detect pathogens at the levels tested, there were several noteworthy differences. The Roche-454 Titanium platform produced consistently longer reads, even when compared with the latest chemistry updates for the PGM platform. The MiSeq platform produced consistently greater depth and breadth of coverage, while the Ion Torrent was unequaled for speed of sequencing. None of the platforms were able to verify a single nucleotide polymorphism responsible for antiviral resistance in an Influenza A strain isolated from the 2009 H1N1 pandemic. Overall, the benchtop platforms perform well for identification of pathogens from a representative clinical sample. However, unlike identification, characterization of pathogens is likely to require higher titers, multiple libraries and/or multiple sequencing runs.

Project description:BACKGROUND: We have developed a simulation approach to help determine the optimal mixture of sequencing methods for most complete and cost effective transcriptome sequencing. We compared simulation results for traditional capillary sequencing with "Next Generation" (NG) ultra high-throughput technologies. The simulation model was parameterized using mappings of 130,000 cDNA sequence reads to the Arabidopsis genome (NCBI Accession SRA008180.19). We also generated 454-GS20 sequences and de novo assemblies for the basal eudicot California poppy (Eschscholzia californica) and the magnoliid avocado (Persea americana) using a variety of methods for cDNA synthesis. RESULTS: The Arabidopsis reads tagged more than 15,000 genes, including new splice variants and extended UTR regions. Of the total 134,791 reads (13.8 MB), 119,518 (88.7%) mapped exactly to known exons, while 1,117 (0.8%) mapped to introns, 11,524 (8.6%) spanned annotated intron/exon boundaries, and 3,066 (2.3%) extended beyond the end of annotated UTRs. Sequence-based inference of relative gene expression levels correlated significantly with microarray data. As expected, NG sequencing of normalized libraries tagged more genes than non-normalized libraries, although non-normalized libraries yielded more full-length cDNA sequences. The Arabidopsis data were used to simulate additional rounds of NG and traditional EST sequencing, and various combinations of each. Our simulations suggest a combination of FLX and Solexa sequencing for optimal transcriptome coverage at modest cost. We have also developed ESTcalc http://fgp.huck.psu.edu/NG_Sims/ngsim.pl, an online webtool, which allows users to explore the results of this study by specifying individualized costs and sequencing characteristics. CONCLUSION: NG sequencing technologies are a highly flexible set of platforms that can be scaled to suit different project goals. In terms of sequence coverage alone, the NG sequencing is a dramatic advance over capillary-based sequencing, but NG sequencing also presents significant challenges in assembly and sequence accuracy due to short read lengths, method-specific sequencing errors, and the absence of physical clones. These problems may be overcome by hybrid sequencing strategies using a mixture of sequencing methodologies, by new assemblers, and by sequencing more deeply. Sequencing and microarray outcomes from multiple experiments suggest that our simulator will be useful for guiding NG transcriptome sequencing projects in a wide range of organisms.

Dataset Information

Analysis and comparison of the wolf microbiome under different environmental factors using three different data of Next Generation Sequencing.

Publications

Analysis and comparison of the wolf microbiome under different environmental factors using three different data of Next Generation Sequencing.

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