Species Identification and Profiling of Complex Microbial Communities Using Shotgun Illumina Sequencing of 16S rRNA Amplicon Sequences

The high throughput and cost-effectiveness afforded by short-read sequencing technologies, in principle, enable researchers to perform 16S rRNA profiling of complex microbial communities at unprecedented depth and resolution. Existing Illumina sequencing protocols are, however, limited by the fraction of the 16S rRNA gene that is interrogated and therefore limit the resolution and quality of the profiling. To address this, we present the design of a novel protocol for shotgun Illumina sequencing of the bacterial 16S rRNA gene, optimized to capture more than 90% of sequences in the Greengenes database and with nearly twice the resolution of existing protocols. Using several in silico and experimental datasets, we demonstrate that despite the presence of multiple variable and conserved regions, the resulting shotgun sequences can be used to accurately quantify the diversity of complex microbial communities. The reconstruction of a significant fraction of the 16S rRNA gene also enabled high precision (>90%) in species-level identification thereby opening up potential application of this approach for clinical microbial characterization.Comment: 17 pages, 2 tables, 2 figures, supplementary materia

Lipid anti-lipid antibody responses correlate with disease activity in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disorder characterized by broad clinical manifestations including cardiovascular and renal complications with periodic disease flares and significant morbidity and mortality. One of the main contributing factors to the pathology of SLE is the accumulation and impaired clearance of immune complexes of which the principle components are host auto-antigens and antibodies. The contribution of host lipids to the formation of these autoimmune complexes remains poorly defined. The aim of the present study was to identify and analyze candidate lipid autoantigens and their corresponding anti-lipid antibody responses in a well-defined SLE patient cohort using a combination of immunological and biophysical techniques. Disease monitoring in the SLE cohort was undertaken with serial British Isles Lupus Assessment Group (BILAG) scoring. Correlations between specific lipid/anti-lipid responses were investigated as disease activity developed from active flares to quiescent during a follow up period. We report a significant negative correlation between anti-lipid antibodies for 24S-hydroxycholesterol, cardiolipin and phosphatidylserine with SLE disease activity. Taken together, these data suggest that lipid autoantigens represent a new family of biomarkers that can be employed to monitor disease activity plus the efficacy of therapeutic intervention in SLE

Human pharyngeal microbiota in age-related macular degeneration.

BACKGROUND: While the aetiology of age-related macular degeneration (AMD)-a major blinding disease-remains unknown, the disease is strongly associated with variants in the complement factor H (CFH) gene. CFH variants also confer susceptibility to invasive infection with several bacterial colonizers of the nasopharyngeal mucosa. This shared susceptibility locus implicates complement deregulation as a common disease mechanism, and suggests the possibility that microbial interactions with host complement may trigger AMD. In this study, we address this possibility by testing the hypothesis that AMD is associated with specific microbial colonization of the human nasopharynx. RESULTS: High-throughput Illumina sequencing of the V3-V6 region of the microbial 16S ribosomal RNA gene was used to comprehensively and accurately describe the human pharyngeal microbiome, at genus level, in 245 AMD patients and 386 controls. Based on mean and differential microbial abundance analyses, we determined an overview of the pharyngeal microbiota, as well as candidate genera (Prevotella and Gemella) suggesting an association towards AMD health and disease conditions. CONCLUSIONS: Utilizing an extensive study population from Singapore, our results provided an accurate description of the pharyngeal microbiota profiles in AMD health and disease conditions. Through identification of candidate genera that are different between conditions, we provide preliminary evidence for the existence of microbial triggers for AMD. Ethical approval for this study was obtained through the Singapore Health Clinical Institutional Review Board, reference numbers R799/63/2010 and 2010/585/A

Microbiome in the hair follicle of androgenetic alopecia patients.

Androgenetic alopecia is the most common form of hair loss in males. It is a multifactorial condition involving genetic predisposition and hormonal changes. The role of microflora during hair loss remains to be understood. We therefore analyzed the microbiome of hair follicles from hair loss patients and the healthy. Hair follicles were extracted from occipital and vertex region of hair loss patients and healthy volunteers and further dissected into middle and lower compartments. The microbiome was then characterized by 16S rRNA sequencing. Distinct microbial population were found in the middle and lower compartment of hair follicles. Middle hair compartment was predominated by Burkholderia spp. and less diverse; while higher bacterial diversity was observed in the lower hair portion. Occipital and vertex hair follicles did not show significant differences. In hair loss patients, miniaturized vertex hair houses elevated Propionibacterium acnes in the middle and lower compartments while non-miniaturized hair of other regions were comparable to the healthy. Increased abundance of P. acnes in miniaturized hair follicles could be associated to elevated immune response gene expression in the hair follicle

Lipid anti-lipid antibody responses correlate with disease activity in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disorder characterized by broad clinical manifestations including cardiovascular and renal complications with periodic disease flares and significant morbidity and mortality. One of the main contributing factors to the pathology of SLE is the accumulation and impaired clearance of immune complexes of which the principle components are host auto-antigens and antibodies. The contribution of host lipids to the formation of these autoimmune complexes remains poorly defined. The aim of the present study was to identify and analyze candidate lipid autoantigens and their corresponding anti-lipid antibody responses in a well-defined SLE patient cohort using a combination of immunological and biophysical techniques. Disease monitoring in the SLE cohort was undertaken with serial British Isles Lupus Assessment Group (BILAG) scoring. Correlations between specific lipid/anti-lipid responses were investigated as disease activity developed from active flares to quiescent during a follow up period. We report a significant negative correlation between anti-lipid antibodies for 24S-hydroxycholesterol, cardiolipin and phosphatidylserine with SLE disease activity. Taken together, these data suggest that lipid autoantigens represent a new family of biomarkers that can be employed to monitor disease activity plus the efficacy of therapeutic intervention in SLE

Species- and genus-level resolution of various sequencing approaches.

<p>Resolution was measured by the number of OTUs/clusters produced using UCLUST <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060811#pone.0060811-Edgar1" target="_blank">[21]</a> at the species (97% identity) and genus level (95% identity) for 16S rRNA sequences in the Greengenes database, based on various end-sequencing (76 bases in length from either the 5′ or 3′ end) and shotgun-sequencing approaches and primer combinations. A higher OTU/cluster number indicates a theoretical higher level of resolution for taxonomic classification. The numbers in parenthesis provide the purity of clusters as measured by the percentage of clusters with homogenous taxonomy assignments in Greengenes. Entries with the highest resolution and/or purity for each sequencing approach are marked in bold. The primer sequences can be found in <b>Table S4</b> in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060811#pone.0060811.s001" target="_blank">File S1</a></b>.</p

Evaluation of EMIRGE, modQIIME and RTAX on different datasets.

<p>Precision and recall rates for the “Oral”, “Gut”, “Complex” and ABC33 datasets using EMIRGE, modQIIME and RTAX at a 0.1% relative abundance threshold. The percentage of sequences/OTUs removed because of the abundance threshold is given in parentheses for each method.</p

<i>In silico</i> evaluation of 16S rRNA PCR primers.

<p>A) Percentage of sequences matching individual primers, with the top two primers highlighted in boxes. B) Percentage of sequences amplifiable by various primer pairs (338F*/1061R is the best pair). Percentage of matched sequences is measured against the Greengenes 16S rRNA sequence database. See Table S4 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060811#pone.0060811.s001" target="_blank">File S1</a> for primer sequences and results measured against the RDP and SILVA databases. Primer numbering is based on the <i>E. coli</i> system of nomenclature as in Brosius <i>et al</i>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060811#pone.0060811-Brosius1" target="_blank">[37]</a> and for simplicity the same name (say 784F) is used for both forward and reverse primers at a given position.</p

Community composition based on 16S rRNA sequence reconstruction using EMIRGE.

<p>A) Correlation between known and estimated relative abundances of predicted species on three <i>in silico</i> datasets. A log-scaled version of this plot can be seen in <b>Figure S1</b> in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060811#pone.0060811.s001" target="_blank">File S1</a></b>. B) Composition at the phylum level for the throat swab and stool sequencing datasets.</p

Manipulation of microbiota reveals altered callosal myelination and white matter plasticity in a model of Huntington disease

Structural and molecular myelination deficits represent early pathological features of Huntington disease (HD). Recent evidence from germ-free (GF) animals suggests a role for microbiota-gut-brain bidirectional communication in the regulation of myelination. In this study, we aimed to investigate the impact of microbiota on myelin plasticity and oligodendroglial population dynamics in the mixed-sex BACHD mouse model of HD. Ultrastructural analysis of myelin in the corpus callosum revealed alterations of myelin thickness in BACHD GF compared to specific-pathogen free (SPF) mice, whereas no differences were observed between wild-type (WT) groups. In contrast, myelin compaction was altered in all groups when compared to WT SPF animals. Levels of myelin-related proteins were generally reduced, and the number of mature oligodendrocytes was decreased in the prefrontal cortex under GF compared to SPF conditions, regardless of genotype. Minor differences in commensal bacteria at the family and genera levels were found in the gut microbiota of BACHD and WT animals housed in standard living conditions. Our findings indicate complex effects of a germ-free status on myelin-related characteristics, and highlight the adaptive properties of myelination as a result of environmental manipulation.Agency for Science, Technology and Research (A*STAR)Nanyang Technological UniversityPublished versionM.A.P. was supported by funds from theAgency for Science Technology and Research and the NationalUniversity of Singapore. S.P. was supported by funds from LKC Schoolof Medicine and SCELSE