Microbial lineages in sarcoidosis. A metagenomic analysis tailored for low–microbial content samples

RATIONALE: The etiology of sarcoidosis is unknown, but microbial agents are suspected as triggers. OBJECTIVES: We sought to identify bacterial, fungal, or viral lineages in specimens from patients with sarcoidosis enriched relative to control subjects using metagenomic DNA sequencing. Because DNA from environmental contamination contributes disproportionately to samples with low authentic microbial content, we developed improved methods for filtering environmental contamination. METHODS: We analyzed specimens from subjects with sarcoidosis (n = 93), control subjects without sarcoidosis (n = 72), and various environmental controls (n = 150). Sarcoidosis specimens consisted of two independent sets of formalin-fixed, paraffin-embedded lymph node biopsies, BAL, Kveim reagent, and fresh granulomatous spleen from a patient with sarcoidosis. All specimens were analyzed by bacterial 16S and fungal internal transcribed spacer ribosomal RNA gene sequencing. In addition, BAL was analyzed by shotgun sequencing of fractions enriched for viral particles, and Kveim and spleen were subjected to whole-genome shotgun sequencing. MEASUREMENTS AND MAIN RESULTS: In one tissue set, fungi in the Cladosporiaceae family were enriched in sarcoidosis compared with nonsarcoidosis tissues; in the other tissue set, we detected enrichment of several bacterial lineages in sarcoidosis but not Cladosporiaceae. BAL showed limited enrichment of Aspergillus fungi. Several microbial lineages were detected in Kveim and spleen, including Cladosporium. No microbial lineage was enriched in more than one sample type after correction for multiple comparisons. CONCLUSIONS: Metagenomic sequencing revealed enrichment of microbes in single types of sarcoidosis samples but limited concordance across sample types. Statistical analysis accounting for environmental contamination was essential to avoiding false positives

Development Of Introgression Lines And Validation Of Quantitative Trait Loci (Qtl) Conferring Transgressive Variation For Yield In Rice

A set of introgression lines (ILs) were developed in an advanced backcross population from a cross between the donor, O. rufipogon (IRGC 105491) in the genetic background of an elite U.S. variety, cv Jefferson, to confirm the performance of six yield-enhancing QTLs identified from a previous study. At the BC3F4 generation, 50 lines containing the donor alleles at the target QTL regions and the least number of background introgressions were selected for evaluation in standard yield plots at four locations in southern U.S. rice growing regions. The top performing lines were genotyped with fixed SNP assays to provide a higher resolution definition of the size and location of donor introgressions in each IL. Based only on genotypic data, 1-2 plants/family were simultaneously selected for an additional backcrossing to Jefferson and screened with SSRs and InDel markers for selecting individuals with minimum background, and for recombination within the target QTL regions as the basis for fine mapping. NIL status was confirmed using the 384 SNP assay and additional genotyping conducted with SNP markers

Additional file 1: Figure S1. of Composition and dynamics of the respiratory tract microbiome in intubated patients

Comparison of beta diversity among intubated subjects and healthy controls. Principal coordinate analysis was performed on pairwise weighted Jaccard distances for samples from intubated subjects (blue) and healthy control subjects (red), based on sequence read counts aggregated at family-level taxonomy (as in Fig. 2). Panel A depicts upper respiratory tract samples. Panel B depicts lower respiratory tract samples. The proportion of variance explained by each principal coordinate is noted parenthetically along the horizontal and vertical axes. (PDF 45.8 KB

Additional file 3: of Composition and dynamics of the respiratory tract microbiome in intubated patients

Closed-reference OTU table. Closed-reference OTU taxonomic assignments. Sequence data was analyzed using the Quantitative Insights Into Microbial Ecology (QIIME) bioinformatics pipeline, version 1.8.0. Sequence alignment was performed via PyNAST, closed-reference operational taxonomic unit (OTU) formation, and taxonomic assignment was based on the Greengenes (13.8) taxonomy. Closed-reference OTUs were used in all comparisons between intubated subjects and healthy controls. (ZIP 90.8 KB

Molecular analysis of the endobronchial stent microbial biofilm reveals bacterial communities that associate with stent material and frequent fungal constituents.

Endobronchial stents are increasingly used to treat airway complications in multiple conditions including lung transplantation but little is known about the biofilms that form on these devices. We applied deep sequencing to profile luminal biofilms of 46 endobronchial stents removed from 20 subjects primarily with lung transplantation-associated airway compromise. Microbial communities were analyzed by bacterial 16S rRNA and fungal ITS marker gene sequencing. Corynebacterium was the most common bacterial taxa across biofilm communities. Clustering analysis revealed three bacterial biofilm types: one low diversity and dominated by Corynebacterium; another was polymicrobial and characterized by Staphylococcus; and the third was polymicrobial and associated with Pseudomonas, Streptococcus, and Prevotella. Biofilm type was significantly correlated with stent material: covered metal with the Staphylococcus-type biofilm, silicone with the Corynebacterium-dominated biofilm, and uncovered metal with the polymicrobial biofilm. Subjects with sequential stents had frequent transitions between community types. Fungal analysis found Candida was most prevalent, Aspergillus was common and highly enriched in two of three stents associated with airway anastomotic dehiscence, and fungal taxa not typically considered pathogens were highly enriched in some stents. Thus, molecular analysis revealed a complex and dynamic endobronchial stent biofilm with three bacterial types that associate with stent material, a central role for Corynebacterium, and that both expected and unexpected fungi inhabit this unique niche. The current work provides a foundation for studies to investigate the relationship between stent biofilm composition and clinical outcomes, mechanisms of biofilm establishment, and strategies for improved stent technology and use in airway compromise