Dataset Information

High-Resolution Profiling of Gut Bacterial Communities in an Invasive Beetle using PacBio SMRT Sequencing System.

ABSTRACT: Dendroctonus valens, an invasive bark beetle, has caused severe damage to Chinese forests. Previous studies have highlighted the importance of the gut microbiota and its fundamental role in host fitness. Culture-dependent and culture-independent methods have been applied in analyzing beetles' gut microbiota. The former method cannot present a whole picture of the community, and the latter mostly generates short read lengths that cannot be assigned to species. Here, the PacBio sequencing system was utilized to capture full-length 16S rRNA sequences in D. valens gut throughout its ontogeny. A total of eight phyla, 55 families, 102 genera, and 253 species were identified. Bacterial communities in colonized beetles have the greatest richness but the lowest evenness in all life stages, which is different from those in young larvae. Pseudomonas sp., Serratia liquefaciens possess high abundance throughout its ontogeny and may serve as members of the core bacteriome. A phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) analysis predicted that gut microbiota in larvae are rich in genes involved in carbohydrate, energy metabolism. Gut microbiota in both larvae and colonized beetles are rich in xenobiotics and terpenoids biodegradation, which are decreased in dispersal beetles. Considering that the results are based mainly on the analysis of 16S rRNA sequencing and PICRUSt prediction, further confirmation is needed to improve the knowledge of the gut microbiota in D. valens and help to resolve taxonomic uncertainty at the species level.

SUBMITTER: Xu L

PROVIDER: S-EPMC6722854 | biostudies-other | 2019 Aug

REPOSITORIES: biostudies-other

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High-Resolution Profiling of Gut Bacterial Communities in an Invasive Beetle using PacBio SMRT Sequencing System.

Xu Letian L Sun Liuwei L Zhang Shihan S Wang Shanshan S Lu Min M

Insects 20190814 8

<i>Dendroctonus valens</i>, an invasive bark beetle, has caused severe damage to Chinese forests. Previous studies have highlighted the importance of the gut microbiota and its fundamental role in host fitness. Culture-dependent and culture-independent methods have been applied in analyzing beetles' gut microbiota. The former method cannot present a whole picture of the community, and the latter mostly generates short read lengths that cannot be assigned to species. Here, the PacBio sequencing s ...[more]

PMID: 31416137

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Project description:BackgroundAn approach utilizing the long-read capability of the Oxford Nanopore MinION to rapidly sequence bacterial ribosomal operons of complex natural communities was developed. Microbial fingerprinting employs domain-specific forward primers (16S rRNA subunit), reverse primers (23S rRNA subunit), and a high-fidelity Taq polymerase with proofreading capabilities. Amplicons contained both ribosomal subunits for broad-based phylogenetic assignment (~ 3900 bp of sequence), plus the intergenic spacer (ITS) region (~ 300 bp) for potential strain-specific identification.ResultsTo test the approach, bacterial rRNA operons (~ 4200 bp) were amplified from six DNA samples employing a mixture of farm soil and bioreactor DNA in known concentrations. Each DNA sample mixture was barcoded, sequenced in quadruplicate (n = 24), on two separate 6-h runs using the MinION system (R7.3 flow cell; MAP005 and 006 chemistry). From nearly 90,000 MinION reads, roughly 33,000 forward and reverse sequences were obtained. This yielded over 10,000 2D sequences which were analyzed using a simplified data analysis pipeline based on NCBI Blast and assembly with Geneious software. The method could detect over 1000 operational taxonomic units in the sample sets in a quantitative manner. Global sequence coverage for the various rRNA operons ranged from 1 to 1951x. An iterative assembly scheme was developed to reconstruct those rRNA operons with > 35x coverage from a set of 30 operational taxonomic units (OTUs) among the Proteobacteria, Actinobacteria, Acidobacteria, Firmicutes, and Gemmatimonadetes. Phylogenetic analysis of the 16S rRNA and 23S rRNA genes from each operon demonstrated similar tree topologies with species/strain-level resolution.ConclusionsThis sequencing method represents a cost-effective way to profile microbial communities. Because the MinION is small, portable, and runs on a laptop, the possibility of microbiota characterization in the field or on robotic platforms becomes realistic.

Dataset Information

High-Resolution Profiling of Gut Bacterial Communities in an Invasive Beetle using PacBio SMRT Sequencing System.

Publications

High-Resolution Profiling of Gut Bacterial Communities in an Invasive Beetle using PacBio SMRT Sequencing System.

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