Reducing the Effects of PCR Amplification and Sequencing Artifacts on 16S rRNA-Based Studies

The advent of next generation sequencing has coincided with a growth in interest in using these approaches to better understand the role of the structure and function of the microbial communities in human, animal, and environmental health. Yet, use of next generation sequencing to perform 16S rRNA gene sequence surveys has resulted in considerable controversy surrounding the effects of sequencing errors on downstream analyses. We analyzed 2.7×10[superscript 6] reads distributed among 90 identical mock community samples, which were collections of genomic DNA from 21 different species with known 16S rRNA gene sequences; we observed an average error rate of 0.0060. To improve this error rate, we evaluated numerous methods of identifying bad sequence reads, identifying regions within reads of poor quality, and correcting base calls and were able to reduce the overall error rate to 0.0002. Implementation of the PyroNoise algorithm provided the best combination of error rate, sequence length, and number of sequences. Perhaps more problematic than sequencing errors was the presence of chimeras generated during PCR. Because we knew the true sequences within the mock community and the chimeras they could form, we identified 8% of the raw sequence reads as chimeric. After quality filtering the raw sequences and using the Uchime chimera detection program, the overall chimera rate decreased to 1%. The chimeras that could not be detected were largely responsible for the identification of spurious operational taxonomic units (OTUs) and genus-level phylotypes. The number of spurious OTUs and phylotypes increased with sequencing effort indicating that comparison of communities should be made using an equal number of sequences. Finally, we applied our improved quality-filtering pipeline to several benchmarking studies and observed that even with our stringent data curation pipeline, biases in the data generation pipeline and batch effects were observed that could potentially confound the interpretation of microbial community data.National Institutes of Health (U.S.) (1R01HG005975-01)National Science Foundation (U.S.) (award #0743432)National Institutes of Health (U.S.) (grant NIHU54HG004969

Books

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Composition of the Adult Digestive Tract Bacterial Microbiome Based on Seven Mouth Surfaces, Tonsils, Throat and Stool Samples

Background: To understand the relationship between our bacterial microbiome and health, it is essential to define the microbiome in the absence of disease. The digestive tract includes diverse habitats and hosts the human body's greatest bacterial density. We describe the bacterial community composition of ten digestive tract sites from more than 200 normal adults enrolled in the Human Microbiome Project, and metagenomically determined metabolic potentials of four representative sites. Results: The microbiota of these diverse habitats formed four groups based on similar community compositions: buccal mucosa, keratinized gingiva, hard palate; saliva, tongue, tonsils, throat; sub- and supra-gingival plaques; and stool. Phyla initially identified from environmental samples were detected throughout this population, primarily TM7, SR1, and Synergistetes. Genera with pathogenic members were well-represented among this disease-free cohort. Tooth-associated communities were distinct, but not entirely dissimilar, from other oral surfaces. The Porphyromonadaceae, Veillonellaceae and Lachnospiraceae families were common to all sites, but the distributions of their genera varied significantly. Most metabolic processes were distributed widely throughout the digestive tract microbiota, with variations in metagenomic abundance between body habitats. These included shifts in sugar transporter types between the supragingival plaque, other oral surfaces, and stool; hydrogen and hydrogen sulfide production were also differentially distributed. Conclusions: The microbiomes of ten digestive tract sites separated into four types based on composition. A core set of metabolic pathways was present across these diverse digestive tract habitats. These data provide a critical baseline for future studies investigating local and systemic diseases affecting human health

On the shape of H2 performance criteria

An H2 control performance criterion dependent on a controller parameter vector is analyzed from the point of view of evaluating the domain of attraction of its global minimum when a gradient-based algorithm is used in tuning the parameters. The objective of this analysis is twofold: (i) examine how some design parameters of the criterion can be used to enlarge the domain of attraction; (ii) examine how the minimization of a sequence of intermediate criteria can possibly lead to the global minimum of the original criterion without the danger of entrapment in a local minimum

Ion-radical synergy in HfO2 etching studied with a XeF2/Ar+ beam setup

To gain more insight into fundamental aspects of the etching behavior of Hf-based high-k materials in plasma etch reactors, HfO2 films were etched in a multiple-beam setup consisting of a low energy Ar+ ion beam and a XeF2 radical beam. The etch rate and etch products were monitored by real-time ellipsometry and mass spectrometry, resp. Although etching of HfO2 in XeF2/Ar+ chem. is mainly a phys. effect, an unambiguous proof of the ion-radical synergistic effect for the etching of HfO2 is presented. The etch yield for 400 eV Ar+ ions at a substrate temp. of 300 DegC was 0.3 atoms/ion for Ar+ sputtering and increased to 2 atoms/ion when XeF2 was also supplied. The etch yield proved to follow the common square root of ion energy dependence both for pure sputtering and radical enhanced etching, with a threshold energy at room temp. of 69+-17 eV for Ar+ ions and 54+-14 eV for Ar+ ions with XeF2

The Treatment-Naive Microbiome in New-Onset Crohn\u27s Disease

Inflammatory bowel diseases (IBDs), including Crohn\u27s disease (CD), are genetically linked to host pathways that implicate an underlying role for aberrant immune responses to intestinal microbiota. However, patterns of gut microbiome dysbiosis in IBD patients are inconsistent among published studies. Using samples from multiple gastrointestinal locations collected prior to treatment in new-onset cases, we studied the microbiome in the largest pediatric CD cohort to date. An axis defined by an increased abundance in bacteria which include Enterobacteriaceae, Pasteurellacaea, Veillonellaceae, and Fusobacteriaceae, and decreased abundance in Erysipelotrichales, Bacteroidales, and Clostridiales, correlates strongly with disease status. Microbiome comparison between CD patients with and without antibiotic exposure indicates that antibiotic use amplifies the microbial dysbiosis associated with CD. Comparing the microbial signatures between the ileum, the rectum, and fecal samples indicates that at this early stage of disease, assessing the rectal mucosal-associated microbiome offers unique potential for convenient and early diagnosis of CD

A direct comparison of the KB™ Basecaller and phred for identifying the bases from DNA sequencing using chain termination chemistry

<p>Abstract</p> <p>Background</p> <p>Relatively recently, the software KB™ Basecaller has replaced <it>phred </it>for identifying the bases from raw sequence data in DNA sequencing employing dideoxy chemistry. We have measured quantitatively the consequences of that change.</p> <p>Results</p> <p>The high quality sequence segment of reads derived from the KB™ Basecaller were, on average, 30-to-50 bases longer than reads derived from <it>phred</it>. However, microbe identification appeared to have been unaffected by the change in software.</p> <p>Conclusions</p> <p>We have demonstrated a modest, but statistically significant, superiority in high quality read length of the KB™ Basecaller compared to <it>phred</it>. We found no statistically significant difference between the numbers of microbial species identified from the sequence data.</p