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September 22, 2019  |  

Using PacBio long-read high-throughput microbial gene amplicon sequencing to evaluate infant formula safety.

Infant formula (IF) requires a strict microbiological standard because of the high vulnerability of infants to foodborne diseases. The current study used the PacBio single molecule real-time (SMRT) sequencing platform to generate full-length 16S rRNA-based bacterial microbiota profiles of thirty Chinese domestic and imported IF samples. A total of 600 species were identified, dominated by Streptococcus thermophilus, Lactococcus lactis and Lactococcus piscium. Distinctive bacterial profiles were observed between the two sample groups, as confirmed with both principal coordinate analysis and multivariate analysis of variance. Moreover, the product whey protein nitrogen index (WPNI), representing the degree of preheating, negatively correlated with the relative abundances of the Bacillus genus. Our study has demonstrated the application of the PacBio SMRT sequencing platform in assessing the bacterial contamination of IF products, which is of interest to the dairy industry for effective monitoring of microbial quality and safety during production.


September 22, 2019  |  

Evaluation of PacBio sequencing for full-length bacterial 16S rRNA gene classification.

Currently, bacterial 16S rRNA gene analyses are based on sequencing of individual variable regions of the 16S rRNA gene (Kozich, et al Appl Environ Microbiol 79:5112-5120, 2013).This short read approach can introduce biases. Thus, full-length bacterial 16S rRNA gene sequencing is needed to reduced biases. A new alternative for full-length bacterial 16S rRNA gene sequencing is offered by PacBio single molecule, real-time (SMRT) technology. The aim of our study was to validate PacBio P6 sequencing chemistry using three approaches: 1) sequencing the full-length bacterial 16S rRNA gene from a single bacterial species Staphylococcus aureus to analyze error modes and to optimize the bioinformatics pipeline; 2) sequencing the full-length bacterial 16S rRNA gene from a pool of 50 different bacterial colonies from human stool samples to compare with full-length bacterial 16S rRNA capillary sequence; and 3) sequencing the full-length bacterial 16S rRNA genes from 11 vaginal microbiome samples and compare with in silico selected bacterial 16S rRNA V1V2 gene region and with bacterial 16S rRNA V1V2 gene regions sequenced using the Illumina MiSeq.Our optimized bioinformatics pipeline for PacBio sequence analysis was able to achieve an error rate of 0.007% on the Staphylococcus aureus full-length 16S rRNA gene. Capillary sequencing of the full-length bacterial 16S rRNA gene from the pool of 50 colonies from stool identified 40 bacterial species of which up to 80% could be identified by PacBio full-length bacterial 16S rRNA gene sequencing. Analysis of the human vaginal microbiome using the bacterial 16S rRNA V1V2 gene region on MiSeq generated 129 operational taxonomic units (OTUs) from which 70 species could be identified. For the PacBio, 36,000 sequences from over 58,000 raw reads could be assigned to a barcode, and the in silico selected bacterial 16S rRNA V1V2 gene region generated 154 OTUs grouped into 63 species, of which 62% were shared with the MiSeq dataset. The PacBio full-length bacterial 16S rRNA gene datasets generated 261 OTUs, which were grouped into 52 species, of which 54% were shared with the MiSeq dataset. Alpha diversity index reported a higher diversity in the MiSeq dataset.The PacBio sequencing error rate is now in the same range of the previously widely used Roche 454 sequencing platform and current MiSeq platform. Species-level microbiome analysis revealed some inconsistencies between the full-length bacterial 16S rRNA gene capillary sequencing and PacBio sequencing.


September 22, 2019  |  

Long-read, Single Molecule, Real-Time (SMRT) DNA Sequencing for metagenomic applications

In this chapter, we describe applications of single molecule, real-time (SMRT) DNA sequencing toward metagenomic research. The long sequence reads, combined with a lack of bias with respect to DNA sequence context or GC content, facilitate a more comprehensive analysis of the genomic constitution of microbial communities. Full-length 16S RNA gene sequencing at high (>99%) accuracy allows for species-level characterization of community members concomitant with the determination of community structure. The application of SMRT sequencing to whole-community shotgun microbial metagenomics has also been discussed.


September 22, 2019  |  

Metataxonomics reveal vultures as a reservoir for Clostridium perfringens.

The Old World vulture may carry and spread pathogens for emerging infections since they feed on the carcasses of dead animals and participate in the sky burials of humans, some of whom have died from communicable diseases. Therefore, we studied the precise fecal microbiome of the Old World vulture with metataxonomics, integrating the high-throughput sequencing of almost full-length small subunit ribosomal RNA (16S rRNA) gene amplicons in tandem with the operational phylogenetic unit (OPU) analysis strategy. Nine vultures of three species were sampled using rectal swabs on the Qinghai-Tibet Plateau, China. Using the Pacific Biosciences sequencing platform, we obtained 54 135 high-quality reads of 16S rRNA amplicons with an average of 1442±6.9?bp in length and 6015±1058 reads per vulture. Those sequences were classified into 314 OPUs, including 102 known species, 50 yet to be described species and 161 unknown new lineages of uncultured representatives. Forty-five species have been reported to be responsible for human outbreaks or infections, and 23 yet to be described species belong to genera that include pathogenic species. Only six species were common to all vultures. Clostridium perfringens was the most abundant and present in all vultures, accounting for 30.8% of the total reads. Therefore, using the new technology, we found that vultures are an important reservoir for C. perfringens as evidenced by the isolation of 107 strains encoding for virulence genes, representing 45 sequence types. Our study suggests that the soil-related C. perfringens and other pathogens could have a reservoir in vultures and other animals.


September 22, 2019  |  

Single-molecule long-read 16S sequencing to characterize the lung microbiome from mechanically ventilated patients with suspected pneumonia.

In critically ill patients, the development of pneumonia results in significant morbidity and mortality and additional health care costs. The accurate and rapid identification of the microbial pathogens in patients with pulmonary infections might lead to targeted antimicrobial therapy with potentially fewer adverse effects and lower costs. Major advances in next-generation sequencing (NGS) allow culture-independent identification of pathogens. The present study used NGS of essentially full-length PCR-amplified 16S ribosomal DNA from the bronchial aspirates of intubated patients with suspected pneumonia. The results from 61 patients demonstrated that sufficient DNA was obtained from 72% of samples, 44% of which (27 samples) yielded PCR amplimers suitable for NGS. Out of the 27 sequenced samples, only 20 had bacterial culture growth, while the microbiological and NGS identification of bacteria coincided in 17 (85%) of these samples. Despite the lack of bacterial growth in 7 samples that yielded amplimers and were sequenced, the NGS identified a number of bacterial species in these samples. Overall, a significant diversity of bacterial species was identified from the same genus as the predominant cultured pathogens. The numbers of NGS-identifiable bacterial genera were consistently higher than identified by standard microbiological methods. As technical advances reduce the processing and sequencing times, NGS-based methods will ultimately be able to provide clinicians with rapid, precise, culture-independent identification of bacterial, fungal, and viral pathogens and their antimicrobial sensitivity profiles. Copyright © 2014, American Society for Microbiology. All Rights Reserved.


September 22, 2019  |  

Long-term changes of bacterial and viral compositions in the intestine of a recovered Clostridium difficile patient after fecal microbiota transplantation

Fecal microbiota transplantation (FMT) is an effective treatment for recurrent Clostridium difficile infections (RCDIs). However, long-term effects on the patients’ gut microbiota and the role of viruses remain to be elucidated. Here, we characterized bacterial and viral microbiota in the feces of a cured RCDI patient at various time points until 4.5 yr post-FMT compared with the stool donor. Feces were subjected to DNA sequencing to characterize bacteria and double-stranded DNA (dsDNA) viruses including phages. The patient’s microbial communities varied over time and showed little overall similarity to the donor until 7 mo post-FMT, indicating ongoing gut microbiota adaption in this time period. After 4.5 yr, the patient’s bacteria attained donor-like compositions at phylum, class, and order levels with similar bacterial diversity. Differences in the bacterial communities between donor and patient after 4.5 yr were seen at lower taxonomic levels. C. difficile remained undetectable throughout the entire timespan. This demonstrated sustainable donor feces engraftment and verified long-term therapeutic success of FMT on the molecular level. Full engraftment apparently required longer than previously acknowledged, suggesting the implementation of year-long patient follow-up periods into clinical practice. The identified dsDNA viruses were mainly Caudovirales phages. Unexpectedly, sequences related to giant algae–infecting Chlorella viruses were also detected. Our findings indicate that intestinal viruses may be implicated in the establishment of gut microbiota. Therefore, virome analyses should be included in gut microbiota studies to determine the roles of phages and other viruses—such as Chlorella viruses—in human health and disease, particularly during RCDI.


September 22, 2019  |  

Investigating bacterial population structure and dynamics in traditional koumiss from Inner Mongolia using single molecule real-time sequencing.

Koumiss is considered as a complete dairy product high in nutrients and with medicinal properties. The bacterial communities involved in production of koumiss play a crucial role in the fermentation cycle. To reveal bacterial biodiversity in koumiss and the dynamics of succession in bacterial populations during fermentation, 22 samples were collected from 5 sampling sites and the full length of the 16S ribosomal RNA genes sequenced using single molecule real-time sequencing technology. One hundred forty-eight species were identified from 82 bacterial genera and 8 phyla. These results suggested that the structural difference in the bacterial community could be attributed to geographical location. The most significant difference in bacterial composition occurred in samples from group D compared with other groups. The sampling location of group D was distant from the city and maintained the primitive local nomadic life. The dynamics of succession in bacterial communities showed that Lactobacillus helveticus increased in abundance from 0 to 9h and reached its peak at 9h and then decreased. In contrast, Enterococcus faecalis, Enterococcus durans, and Enterococcus casseliflavus increased gradually throughout the fermentation process, and reached a maximum after 24h. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.


September 22, 2019  |  

Evaluation of bacterial contamination in raw milk, ultra-high temperature milk and infant formula using single molecule, real-time sequencing technology.

The Pacific Biosciences (Menlo Park, CA) single molecule, real-time sequencing technology (SMRT) was reported to have some advantages in analyzing the bacterial profile of environmental samples. In this study, the presence of bacterial contaminants in raw milk, UHT milk, and infant formula was determined by SMRT sequencing of the full length 16S rRNA gene. The bacterial profiles obtained at different taxonomic levels revealed clear differences in bacterial community structure across the 16 analyzed dairy samples. No indicative pathogenic bacteria were found in any of these tested samples. However, some of the detected bacterial species (e.g., Bacillus cereus, Enterococcus casseliflavus, and Enterococcus gallinarum) might potentially relate with product quality defects and bacterial antibiotic gene transfer. Although only a limited number of dairy samples were analyzed here, our data have demonstrated for the first time the feasibility of using the SMRT sequencing platform in detecting bacterial contamination. Our paper also provides interesting reference information for future development of new precautionary strategies for controlling the dairy safety in large-scale industrialized production lines. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.


September 22, 2019  |  

High-resolution phylogenetic microbial community profiling.

Over the past decade, high-throughput short-read 16S rRNA gene amplicon sequencing has eclipsed clone-dependent long-read Sanger sequencing for microbial community profiling. The transition to new technologies has provided more quantitative information at the expense of taxonomic resolution with implications for inferring metabolic traits in various ecosystems. We applied single-molecule real-time sequencing for microbial community profiling, generating full-length 16S rRNA gene sequences at high throughput, which we propose to name PhyloTags. We benchmarked and validated this approach using a defined microbial community. When further applied to samples from the water column of meromictic Sakinaw Lake, we show that while community structures at the phylum level are comparable between PhyloTags and Illumina V4 16S rRNA gene sequences (iTags), variance increases with community complexity at greater water depths. PhyloTags moreover allowed less ambiguous classification. Last, a platform-independent comparison of PhyloTags and in silico generated partial 16S rRNA gene sequences demonstrated significant differences in community structure and phylogenetic resolution across multiple taxonomic levels, including a severe underestimation in the abundance of specific microbial genera involved in nitrogen and methane cycling across the Lake’s water column. Thus, PhyloTags provide a reliable adjunct or alternative to cost-effective iTags, enabling more accurate phylogenetic resolution of microbial communities and predictions on their metabolic potential.


September 22, 2019  |  

Different next generation sequencing platforms produce different microbial profiles and diversity in cystic fibrosis sputum.

Cystic fibrosis (CF) is an autosomal recessive disease characterized by recurrent lung infections. Studies of the lung microbiome have shown an association between decreasing diversity and progressive disease. 454 pyrosequencing has frequently been used to study the lung microbiome in CF, but will no longer be supported. We sought to identify the benefits and drawbacks of using two state-of-the-art next generation sequencing (NGS) platforms, MiSeq and PacBio RSII, to characterize the CF lung microbiome. Each has its advantages and limitations.Twelve samples of extracted bacterial DNA were sequenced on both MiSeq and PacBio NGS platforms. DNA was amplified for the V4 region of the 16S rRNA gene and libraries were sequenced on the MiSeq sequencing platform, while the full 16S rRNA gene was sequenced on the PacBio RSII sequencing platform. Raw FASTQ files generated by the MiSeq and PacBio platforms were processed in mothur v1.35.1.There was extreme discordance in alpha-diversity of the CF lung microbiome when using the two platforms. Because of its depth of coverage, sequencing of the 16S rRNA V4 gene region using MiSeq allowed for the observation of many more operational taxonomic units (OTUs) and higher Chao1 and Shannon indices than the PacBio RSII. Interestingly, several patients in our cohort had Escherichia, an unusual pathogen in CF. Also, likely because of its coverage of the complete 16S rRNA gene, only PacBio RSII was able to identify Burkholderia, an important CF pathogen.When comparing microbiome diversity in clinical samples from CF patients using 16S sequences, MiSeq and PacBio NGS platforms may generate different results in microbial community composition and structure. It may be necessary to use different platforms when trying to correctly identify dominant pathogens versus measuring alpha-diversity estimates, and it would be important to use the same platform for comparisons to minimize errors in interpretation. Copyright © 2016 Elsevier B.V. All rights reserved.


September 22, 2019  |  

Evaluation of 16S rRNA amplicon sequencing using two next-generation sequencing technologies for phylogenetic analysis of the rumen bacterial community in steers.

Next generation sequencing technologies have vastly changed the approach of sequencing of the 16S rRNA gene for studies in microbial ecology. Three distinct technologies are available for large-scale 16S sequencing. All three are subject to biases introduced by sequencing error rates, amplification primer selection, and read length, which can affect the apparent microbial community. In this study, we compared short read 16S rRNA variable regions, V1-V3, with that of near-full length 16S regions, V1-V8, using highly diverse steer rumen microbial communities, in order to examine the impact of technology selection on phylogenetic profiles. Short paired-end reads from the Illumina MiSeq platform were used to generate V1-V3 sequence, while long “circular consensus” reads from the Pacific Biosciences RSII instrument were used to generate V1-V8 data. The two platforms revealed similar microbial operational taxonomic units (OTUs), as well as similar species richness, Good’s coverage, and Shannon diversity metrics. However, the V1-V8 amplified ruminal community resulted in significant increases in several orders of taxa, such as phyla Proteobacteria and Verrucomicrobia (P < 0.05). Taxonomic classification accuracy was also greater in the near full-length read. UniFrac distance matrices using jackknifed UPGMA clustering also noted differences between the communities. These data support the consensus that longer reads result in a finer phylogenetic resolution that may not be achieved by shorter 16S rRNA gene fragments. Our work on the cattle rumen bacterial community demonstrates that utilizing near full-length 16S reads may be useful in conducting a more thorough study, or for developing a niche-specific database to use in analyzing data from shorter read technologies when budgetary constraints preclude use of near-full length 16S sequencing. Copyright © 2016 Elsevier B.V. All rights reserved.


September 22, 2019  |  

Bacterial microbiota of Kazakhstan cheese revealed by single molecule real time (SMRT) sequencing and its comparison with Belgian, Kalmykian and Italian artisanal cheeses

In Kazakhstan, traditional artisanal cheeses have a long history and are widely consumed. The unique characteristics of local artisanal cheeses are almost completely preserved. However, their microbial communities have rarely been reported. The current study firstly generated the Single Molecule, Real-Time (SMRT) sequencing bacterial diversity profiles of 6 traditional artisanal cheese samples of Kazakhstan origin, followed by comparatively analyzed the microbiota composition between the current dataset and those from cheeses originated from Belgium, Russian Republic of Kalmykia (Kalmykia) and Italy.


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