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Wednesday, February 26, 2020

Profiling metagenomic communities using circular consensus and Single Molecule, Real-Time Sequencing.

There are many sequencing-based approaches to understanding complex metagenomic communities spanning targeted amplification to whole-sample shotgun sequencing. While targeted approaches provide valuable data at low sequencing depth, they are limited by primer design and PCR amplification. Whole-sample shotgun experiments generally use short-read, second-generation sequencing, which results in data processing difficulties. For example, reads less than 1 kb in length will likely not cover a complete gene or region of interest, and will require assembly. This not only introduces the possibility of incorrectly combining sequence from different community members, it requires a high depth of coverage. As such, rare community members…

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Wednesday, February 26, 2020

Analysis of full-length metagenomic 16S genes by Single Molecule, Real-Time Sequencing

High-throughput sequencing of the complete 16S rRNA gene has become a valuable tool for characterizing microbial communities. However, the short reads produced by second-generation sequencing cannot provide taxonomic classification below the genus level. In this study, we demonstrate the capability of PacBio’s Single Molecule, Real-Time (SMRT) Sequencing to generate community profiles using mock microbial community samples from BEI Resources. We also evaluate multiplexing capabilities using PacBio barcodes on pooled samples comprising heterogeneous 16S amplicon populations representing soil, fecal, and mock communities.

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Wednesday, February 26, 2020

Profiling metagenomic communities using circular consensus and Single Molecule, Real-Time Sequencing

There are many sequencing-based approaches to understanding complex metagenomic communities, spanning targeted amplification to whole-sample shotgun sequencing. While targeted approaches provide valuable data at low sequencing depth, they are limited by primer design and PCR amplification. Whole-sample shotgun experiments require a high depth of coverage. As such, rare community members may not be represented in the resulting assembly. Circular-consensus, Single Molecule, Real-Time (SMRT) Sequencing reads in the 1-2 kb range, with >99% consensus accuracy, can be efficiently generated for low amounts of input DNA, e.g. as little as 10 ng of input DNA sequenced in 4 SMRT Cells can generate…

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Wednesday, February 26, 2020

Profiling the microbiome in fecal microbiota transplantation using circular consensus and Single Molecule, Real-Time Sequencing

There are many sequencing-based approaches to understanding complex metagenomic communities spanning targeted amplification to whole-sample shotgun sequencing. While targeted approaches provide valuable data at low sequencing depth, they are limited by primer design and PCR. Whole-sample shotgun experiments generally use short-read sequencing, which results in data processing difficulties. For example, reads less than 500bp in length will rarely cover a complete gene or region of interest, and will require assembly. This not only introduces the possibility of incorrectly combining sequence from different community members, it requires a high depth of coverage. As such, rare community members may not be represented…

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Wednesday, February 26, 2020

Minimization of chimera formation and substitution errors in full-length 16S PCR amplification

The constituents and intra-communal interactions of microbial populations have garnered increasing interest in areas such as water remediation, agriculture and human health. One popular, efficient method of profiling communities is to amplify and sequence the evolutionarily conserved 16S rRNA sequence. Currently, most targeted amplification focuses on short, hypervariable regions of the 16S sequence. Distinguishing information not spanned by the targeted region is lost and species-level classification is often not possible. SMRT Sequencing easily spans the entire 1.5 kb 16S gene, and in combination with highly-accurate single-molecule sequences, can improve the identification of individual species in a metapopulation. However, when amplifying…

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Wednesday, February 26, 2020

Low-input long-read sequencing for complete microbial genomes and metagenomic community analysis

Microbial genome sequencing can be done quickly, easily, and efficiently with the PacBio sequencing instruments, resulting in complete de novo assemblies. Alternative protocols have been developed to reduce the amount of purified DNA required for SMRT Sequencing, to broaden applicability to lower-abundance samples. If 50-100 ng of microbial DNA is available, a 10-20 kb SMRTbell library can be made. The resulting library can be loaded onto multiple SMRT Cells, yielding more than enough data for complete assembly of microbial genomes using the SMRT Portal assembly program HGAP, plus base modification analysis. The entire process can be done in less than…

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Wednesday, February 26, 2020

Minimization of chimera formation and substitution errors in full-length 16S PCR amplification

The constituents and intra-communal interactions of microbial populations have garnered increasing interest in areas such as water remediation, agriculture and human health. Amplification and sequencing of the evolutionarily conserved 16S rRNA gene is an efficient method of profiling communities. Currently, most targeted amplification focuses on short, hypervariable regions of the 16S sequence. Distinguishing information not spanned by the targeted region is lost, and species-level classification is often not possible. PacBio SMRT Sequencing easily spans the entire 1.5 kb 16S gene in a single read, producing highly accurate single-molecule sequences that can improve the identification of individual species in a metapopulation.However,…

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Wednesday, February 26, 2020

Profiling complex population genomes with highly accurate single molecule reads: cow rumen microbiomes

Determining compositions and functional capabilities of complex populations is often challenging, especially for sequencing technologies with short reads that do not uniquely identify organisms or genes. Long-read sequencing improves the resolution of these mixed communities, but adoption for this application has been limited due to concerns about throughput, cost and accuracy. The recently introduced PacBio Sequel System generates hundreds of thousands of long and highly accurate single-molecule reads per SMRT Cell. We investigated how the Sequel System might increase understanding of metagenomic communities. In the past, focus was largely on taxonomic classification with 16S rRNA sequencing. Recent expansion to WGS…

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Wednesday, February 26, 2020

Profiling complex communities with highly accurate single molecule reads: cow rumen microbiomes

Determining compositions and functional capabilities of complex populations is often challenging, especially for sequencing technologies with short reads that do not uniquely identify organisms or genes. Long-read sequencing improves the resolution of these mixed communities, but adoption for this application has been limited due to concerns about throughput, cost and accuracy. The recently introduced PacBio Sequel System generates hundreds of thousands of long and highly accurate single-molecule reads per SMRT Cell. We investigated how the Sequel System might increase understanding of metagenomic communities. In the past, focus was largely on taxonomic classification with 16S rRNA sequencing. Recent expansion to WGS…

Read More »

Wednesday, October 23, 2019

Dynamics of coral-associated microbiomes during a thermal bleaching event.

Coral-associated microorganisms play an important role in their host fitness and survival. A number of studies have demonstrated connections between thermal tolerance in corals and the type/relative abundance of Symbiodinium they harbor. More recently, the shifts in coral-associated bacterial profiles were also shown to be linked to the patterns of coral heat tolerance. Here, we investigated the dynamics of Porites lutea-associated bacterial and algal communities throughout a natural bleaching event, using full-length 16S rRNA and internal transcribed spacer sequences (ITS) obtained from PacBio circular consensus sequencing. We provided evidence of significant changes in the structure and diversity of coral-associated microbiomes…

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Wednesday, October 23, 2019

High resolution profiling of coral-associated bacterial communities using full-length 16S rRNA sequence data from PacBio SMRT sequencing system.

Coral reefs are a complex ecosystem consisting of coral animals and a vast array of associated symbionts including the dinoflagellate Symbiodinium, fungi, viruses and bacteria. Several studies have highlighted the importance of coral-associated bacteria and their fundamental roles in fitness and survival of the host animal. The scleractinian coral Porites lutea is one of the dominant reef-builders in the Indo-West Pacific. Currently, very little is known about the composition and structure of bacterial communities across P. lutea reefs. The purpose of this study is twofold: to demonstrate the advantages of using PacBio circular consensus sequencing technology in microbial community studies…

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Sunday, September 22, 2019

Assessing quality of Medicago sativa silage by monitoring bacterial composition with single molecule, real-time sequencing technology and various physiological parameters.

The present study applied the PacBio single molecule, real-time sequencing technology (SMRT) in evaluating the quality of silage production. Specifically, we produced four types of Medicago sativa silages by using four different lactic acid bacteria-based additives (AD-I, AD-II, AD-III and AD-IV). We monitored the changes in pH, organic acids (including butyric acid, the ratio of acetic acid/lactic acid, ?-aminobutyric acid, 4-hyroxy benzoic acid and phenyl lactic acid), mycotoxins, and bacterial microbiota during silage fermentation. Our results showed that the use of the additives was beneficial to the silage fermentation by enhancing a general pH and mycotoxin reduction, while increasing the…

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Sunday, September 22, 2019

Improved performance of the PacBio SMRT technology for 16S rDNA sequencing.

Improved sequencing accuracy was obtained with 16S amplicons from environmental samples and a known pure culture when upgraded Pacific Biosciences (PacBio) hardware and enzymes were used for the single molecule, real-time (SMRT) sequencing platform. The new PacBio RS II system with P4/C2 chemistry, when used with previously constructed libraries (Mosher et al., 2013) surpassed the accuracy of Roche/454 pyrosequencing platform. With accurate read lengths of >1400 base pairs, the PacBio system opens up the possibility of identifying microorganisms to the species level in environmental samples. Copyright © 2014 Elsevier B.V. All rights reserved.

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Sunday, September 22, 2019

A novel lactobacilli-based teat disinfectant for improving bacterial communities in the milks of cow teats with subclinical mastitis.

Teat disinfection pre- and post-milking is important for the overall health and hygiene of dairy cows. The objective of this study was to evaluate the efficacy of a novel probiotic lactobacilli-based teat disinfectant based on changes in somatic cell count (SCC) and profiling of the bacterial community. A total of 69 raw milk samples were obtained from eleven Holstein-Friesian dairy cows over 12 days of teat dipping in China. Single molecule, real-time sequencing technology (SMRT) was employed to profile changes in the bacterial community during the cleaning protocol and to compare the efficacy of probiotic lactic acid bacteria (LAB) and…

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