Illumina-based Sequencing Research Articles

Exploring potential bacterial and fungal biocontrol agents transmitted from seeds to sprouts of wheat

Seed-borne endophytes could be transmitted vertically from generation to generation. This vertical transmission should select against pathogenicity and favour mutualism as these endophytes depend entirely on their host for survival and reproduction. To elucidate the vertically transmitted core bacteriome and mycobiome throughout the germination process of wheat seeds, the bacterial and fungal taxa of sprouts, sprouts from sterilized seeds and seeds of healthy wheat plants were analyzed by Illumina-based sequencing. Results suggested that the sprouts from sterilized seeds contained more diverse fungi than those in the seeds and sprouts, nevertheless, the seeds contained more diverse bacteria than those in sprout samples. The wheat seeds and sprouts contained different dominant bacterial and fungal taxa. Bacterial OTUs belong to Erwinia and Rhizobiales were founded in seed and all sprout samples, but the abundance of Erwinia decreased in sprouts. The dominant fungal genus Emericella was detected in seed and all sprout samples. The results suggested that wheat seeds and sprouts might contain core bacteriome and mycobiome during the development from seeds to sprouts and could transmit in the plant tissues of wheat. The core endophytes should illustrate more intimate correlation with host plants than other microbial taxa and the results are helpful for elucidating the plant-microbe interactions and selecting suitable microbial biocontrol agents for wheat production.

Salinity altered root distribution and increased diversity of bacterial communities in the rhizosphere soil of Jerusalem artichoke.

The interaction between roots and bacterial communities in halophytic species is poorly understood. Here, we used Jerusalem artichoke cultivar Nanyu 1 (NY-1) to characterise root distribution patterns and determine diversity and abundance of bacteria in the rhizosphere soil under variable salinity. Root growth was not inhibited within the salinity range 1.2 to 1.9 g salt/kg, but roots were mainly confined to 0–20 cm soil layer vertically and 0–30 cm horizontally from the plant centre. Root concentrations of K+, Na+, Mg2+ and particularly Ca2+ were relatively high under salinity stress. High salinity stress decreased soil invertase and catalase activity. Using a next-generation, Illumina-based sequencing approach, we determined higher diversity of bacteria in the rhizosphere soil at high than low salinity. More than 15,500 valid reads were obtained, and Proteobacteria, Acidobacteria, Bacteroidetes and Actinobacteria predominated in all samples, accounting for >80% of the reads. On a genus level, 636 genera were common to the low and high salinity treatments at 0–5 cm and 5–10 cm depth. The abundance of Steroidobacter and Sphingomonas was significantly decreased by increasing salinity. Higher Shannon and Chao 1 indices with increasing severity of salt stress indicated that high salt stress increased diversity in the bacterial communities.

Pepino (Solanum muricatum) planting increased diversity and abundance of bacterial communities in karst area.

Soil nutrients and microbial communities are the two key factors in revegetation of barren environments. Ecological stoichiometry plays an important role in ecosystem function and limitation, but the relationships between above- and belowground stoichiometry and the bacterial communities in a typical karst region are poorly understood. We used pepino (Solanum muricatum) to examine the stoichiometric traits between soil and foliage, and determine diversity and abundance of bacteria in the karst soil. The soil had a relatively high pH, low fertility, and coarse texture. Foliar N:P ratio and the correlations with soil nitrogen and phosphorus suggested nitrogen limitation. The planting of pepino increased soil urease activity and decreased catalase activity. Higher diversity of bacteria was determined in the pepino rhizosphere than bulk soil using a next-generation, Illumina-based sequencing approach. Proteobacteria, Acidobacteria, Actinobacteria and Bacteroidetes were the dominant phyla in all samples, accounting for more than 80% of the reads. On a genus level, all 625 detected genera were found in all rhizosphere and bulk soils, and 63 genera showed significant differences among samples. Higher Shannon and Chao 1 indices in the rhizosphere than bulk soil indicated that planting of pepino increased diversity and abundance of bacterial communities in karst area.

A New Approach to Analyzing Endophytic Actinobacterial Population in the Roots of Banana Plants (Musa sp., AAA)

AIM: To elucidate the endophytic actinobacterial populations of healthy banana plant roots. METHODS: A surface sterilization protocol was adopted to remove rhizoplane bacterial DNAs and total DNAs of surface-sterilized roots were purified and analyzed by Illumina-based sequencing. RESULTS and CONCLUSION: A total of 2152102 bacterial sequences remained with an average length of 448bp, 31952 different actinobacterial OTUs at the 97% similarity level were obtained, and the dominant families were Nocardioidaceae, PseudoNocardiaceae and Nocardiaceae, which accounted for 56.37%, 14.36% and 9.77%, respectively. At genus level, Pseudonocardia (11.83%), Rhodococcus (9.74%), Nocardioides (3.06%) were the most abundant taxa. The results indicated that surface sterilization approach with formaldehyde (36%) and Illumina-based sequencing is suitable for analysis of endophytic actinobacterial populations in banana roots.

Mapping Protein-DNA Interactions Using ChIP-exo and Illumina-Based Sequencing.

Chromatin immunoprecipitation (ChIP) provides a means of enriching DNA associated with transcription factors, histone modifications, and indeed any other proteins for which suitably characterized antibodies are available. Over the years, sequence detection has progressed from quantitative real-time PCR and Southern blotting to microarrays (ChIP-chip) and now high-throughput sequencing (ChIP-seq). This progression has vastly increased the sequence coverage and data volumes generated. This in turn has enabled informaticians to predict the identity of multi-protein complexes on DNA based on the overrepresentation of sequence motifs in DNA enriched by ChIP with a single antibody against a single protein. In the course of the development of high-throughput sequencing, little has changed in the ChIP methodology until recently. In the last three years, a number of modifications have been made to the ChIP protocol with the goal of enhancing the sensitivity of the method and further reducing the levels of nonspecific background sequences in ChIPped samples. In this chapter, we provide a brief commentary on these methodological changes and describe a detailed ChIP-exo method able to generate narrower peaks and greater peak coverage from ChIPped material.

Transcriptome analysis of Leucaena leucocephala and identification of highly expressed genes in roots and shoots

Leucaena leucocephala (leucaena) is a fast-growing tree legume highly tolerant to various abiotic and biotic stresses. Because of its abilities to withstand high temperature and prolonged drought and to grow as a disease-free plant, it is an interesting model plant to investigate genetics of stress resistance. The high-level stress resistance may be correlated with higher expression of certain genes in the root, which is the primary site for nutrient and water uptake and also infection by soil-borne pathogens. The objectives of this study were to characterize the transcriptome of leucaena and to identify root-specific genes that may be involved in drought tolerance and disease resistance. Transcriptomes of leucaena were analyzed through Illumina-based sequencing and de novo assembly, which generated 62,299 and 61,591 unigenes (≥ 500 bp) from the root and shoot, respectively. Through a 4 x 180,000 microarray analysis, the expression of 10,435 unigenes were compared between the root and shoot. Upregulated sequences in the root were mostly represented by unigenes that were related to secondary metabolism, while in the shoot, upregulated sequences were mostly represented by unigenes that were involved in carbohydrate and lipid metabolism. The unigenes sharing homology with terpenoid biosynthesis genes and a nicotianamine synthase gene were upregulated more than 100-fold in the root, which indicates that these genes may have important roles in high stress tolerance of leucaena. Cataloging of actively transcribed sequences in the root and shoot will lead to identification of genes for drought tolerance and disease resistance in leucaena.

Transcriptome analysis of soiny mullet (Liza haematocheila) spleen in response to Streptococcus dysgalactiae.

Soiny mullet (Liza haematocheila) is becoming an economically important aquaculture mugilid species in China and other Asian countries. However, increasing incidences of bacterial pathogenic diseases has greatly hampered the production of the soiny mullet. Deeper understanding of the soiny mullet immune system and its related genes in response to bacterial infections are necessary for disease control in this species. In this study, the transcriptomic profile of spleen from soiny mullet challenged with Streptococcus dysgalactiae was analyzed by Illumina-based paired-end sequencing method. After assembly, 86,884 unique transcript fragments (unigenes) were assembled, with an average length of 991bp. Approximately 41,795 (48.1%) unigenes were annotated in the nr NCBI database and 57.9% of the unigenes were similar to that of the Nile tilapia. A total of 24,299 unigenes were categorized into three Gene Ontology (GO) categories (molecular function, cellular component and biological process), 13,570 unigenes into 25 functional Clusters of Orthologous Groups of proteins (COG) categories, and 30,547 unigenes were grouped into 258 known pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Following S.dysgalactiae infection, 11,461 differentially expressed unigenes were identified including 4658 up-regulated unigenes and 6803 down-regulated unigenes. Significant enrichment analysis of these differentially expressed unigenes identified major immune related pathways, including the Toll-like receptor, complement and coagulation cascades, T cell receptor signaling pathway and B cell receptor signaling pathway. In addition, 24,813 simple sequence repeats (SSRs) and 127,503 candidate single nucleotide polymorphisms (SNPs) were identified from the mullet spleen transcriptome. To this date, this study has globally analyzed the transcriptome profile from the spleen of L.haematocheila after S.dysgalactiae infection. Therefore, the results of our study contributes to better on the immune system and defense mechanisms of soiny mullet in response to bacterial infection, and provides valuable references for related studies in mugilidae species which currently lack genomic reference.

A Comprehensive RNA Sequencing Analysis of the Adeno-Associated Virus (AAV) Type 2 Transcriptome Reveals Novel AAV Transcripts, Splice Variants, and Derived Proteins.

Adeno-associated virus (AAV) is recognized for its bipartite life cycle with productive replication dependent on coinfection with adenovirus (Ad) and AAV latency being established in the absence of a helper virus. The shift from latent to Ad-dependent AAV replication is mostly regulated at the transcriptional level. The current AAV transcription map displays highly expressed transcripts as found upon coinfection with Ad. So far, AAV transcripts have only been characterized on the plus strand of the AAV single-stranded DNA genome. The AAV minus strand is assumed not to be transcribed. Here, we apply Illumina-based RNA sequencing (RNA-Seq) to characterize the entire AAV2 transcriptome in the absence or presence of Ad. We find known and identify novel AAV transcripts, including additional splice variants, the most abundant of which leads to expression of a novel 18-kDa Rep/VP fusion protein. Furthermore, we identify for the first time transcription on the AAV minus strand with clustered reads upstream of the p5 promoter, confirmed by 5' rapid amplification of cDNA ends and RNase protection assays. The p5 promoter displays considerable activity in both directions, a finding indicative of divergent transcription. Upon infection with AAV alone, low-level transcription of both AAV strands is detectable and is strongly stimulated upon coinfection with Ad. Next-generation sequencing (NGS) allows unbiased genome-wide analyses of transcription profiles, used here for an in depth analysis of the AAV2 transcriptome during latency and productive infection. RNA-Seq analysis led to the discovery of novel AAV transcripts and splice variants, including a derived, novel 18-kDa Rep/VP fusion protein. Unexpectedly, transcription from the AAV minus strand was discovered, indicative of divergent transcription from the p5 promoter. This finding opens the door for novel concepts of the switch between AAV latency and productive replication. In the absence of a suitable animal model to study AAV in vivo, combined in cellulae and in silico studies will help to forward the understanding of the unique, bipartite AAV life cycle.

Nitrogen loss through anaerobic ammonium oxidation in agricultural drainage ditches

Agricultural drainage ditches are a key conduit of nitrogen from agricultural fields to surface water. Denitrification has been proven to be an important N removal pathway in agricultural drainage ditches; however, it is currently unknown about the role of anaerobic ammonium oxidation (anammox). Here, we investigated the N loss through anammox in six different agricultural drainage ditches, located in Nanjing City, Jiangsu Province (China). Stable isotope-labeling experiments confirmed the anammox activity in every examined ditch, and the potential anammox rates ranged from 4.4 to 23.8 nmol N2 g−1 sediment day−1. The anammox process contributed 2.1–18.8 % to sediment dinitrogen gas production, while the remainder would be due to denitrification. It is estimated that a total loss of 6.1–32.9 g N m-2 per year could be attributed to the anammox process in the examined ditches. Illumina-based 16S rRNA gene sequencing confirmed the presence of diverse anammox bacteria in the examined sediments, including Candidatus Brocadia, Candidatus Kuenenia, and Candidatus Anammoxoglobus. Quantitative PCR further confirmed the presence of anammox bacteria, varying between 1.9 × 104 and 1.2 × 105 copies g−1 sediment. The results of our study showed the evidence of anammox process in agricultural drainage ditches and demonstrated that the contribution of anammox to N removal from agricultural drainage ditches may not be negligible.

Evidence for anaerobic ammonium oxidation process in freshwater sediments of aquaculture ponds

The anaerobic ammonium oxidation (anammox) process, which can simultaneously remove ammonium and nitrite, both toxic to aquatic animals, can be very important to the aquaculture industry. Here, the presence and activity of anammox bacteria in the sediments of four different freshwater aquaculture ponds were investigated by using Illumina-based 16S rRNA gene sequencing, quantitative PCR assays and (15)N stable isotope measurements. Different genera of anammox bacteria were detected in the examined pond sediments, including Candidatus Brocadia, Candidatus Kuenenia and Candidatus Anammoxoglobus, with Candidatus Brocadia being the dominant anammox genus. Quantitative PCR of hydrazine synthase genes showed that the abundance of anammox bacteria ranged from 5.6 × 10(4) to 2.1 × 10(5) copies g(-1) sediment in the examined ponds. The potential anammox rates ranged between 3.7 and 19.4 nmol N2 g(-1) sediment day(-1), and the potential denitrification rates varied from 107.1 to 300.3 nmol N2 g(-1) sediment day(-1). The anammox process contributed 1.2-15.3% to sediment dinitrogen gas production, while the remainder would be due to denitrification. It is estimated that a total loss of 2.1-10.9 g N m(-2) per year could be attributed to the anammox process in the examined ponds, suggesting that this process could contribute to nitrogen removal in freshwater aquaculture ponds.

P1.a.013 Effect of HTR7 gene variants on the efficacy of second-generation antipsychotics with 5-HT7R antagonism in Japanese schizophrenia patients

The Manila clam, Ruditapes philippinarum, is one of the most economically important aquatic clams that are harvested on a large scale by the mariculture industry in China. However, increasing reports of bacterial pathogenic diseases have had a negative effect on the aquaculture industry of R. philippinarum. In the present study, the two transcriptome libraries of untreated (termed H) and challenged Vibrio anguillarum (termed HV) hepatopancreas were constructed and sequenced from Manila clam using an Illumina-based paired-end sequencing platform. In total, 75,302,886 and 66,578,976 high-quality clean reads were assembled from 101,080,746 and 99,673,538 raw data points from the two transcriptome libraries described above, respectively. Furthermore, 156,116 unigenes were generated from 210,685 transcripts, with an N50 length of 1125 bp, and from the annotated SwissProt, NR, NT, KO, GO, KOG and KEGG databases. Moreover, a total of 4071 differentially expressed unigenes (HV vs H) were detected, including 903 up-regulated and 3168 down-regulated genes. Among these differentially expressed unigenes, 226 unigenes were annotated using KEGG annotation in 16 immune-related signaling pathways, including Toll-like receptor, NF-kappa B, MAPK, NOD-like receptor, RIG-I-like receptor, and the TNF and chemokine signaling pathways. Finally, 20,341 simple sequence repeats (SSRs) and 214,430 potential single nucleotide polymorphisms (SNPs) were detected from the H and HV transcriptome libraries. In conclusion, these studies identified many candidate immune-related genes and signaling pathways and conducted a comparative analysis of the differentially expressed unigenes from Manila clam hepatopancreas in response to V. anguillarum stimulation. These data laid the foundation for studying the innate immune systems and defense mechanisms in R. philippinarum.

Transcriptome analysis to identify genes for peptides and proteins involved in immunity and reproduction from male accessory glands and ejaculatory duct of Bactrocera dorsalis

In the male reproductive system of insects, the male accessory glands and ejaculatory duct (MAG/ED) are important organs and their primary function is to enhance the fertility of spermatozoa. Proteins secreted by the MAG/ED are also known to induce post-mating changes and immunity responses in the female insect. To understand the gene expression profile in the MAG/ED of the oriental fruit fly Bactrocera dorsalis (Hendel), that is an important pest in fruits, we performed an Illumina-based deep sequencing of mRNA. This yielded 54,577,630 clean reads corresponding to 4.91Gb total nucleotides that were assembled and clustered to 30,669 unigenes (average 645bp). Among them, 20,419 unigenes were functionally annotated to known proteins/peptides in Gene Orthology, Clusters of Orthologous Groups, Kyoto Encyclopedia of Genes and Genomes pathway databases. Typically, many genes were involved in immunity and these included microbial recognition proteins and antimicrobial peptides. Subsequently, the inducible expression of these immunity-related genes was confirmed by qRT-PCR analysis when insects were challenged with immunity-inducible factors, suggesting their function in guaranteeing fertilization success. Besides, we identified some important reproductive genes such as juvenile hormone- and ecdysteroid-related genes in this de novo assembly. In conclusion, this transcriptomic sequencing of B. dorsalis MAG/ED provides insights to facilitate further functional research of reproduction, immunity and molecular evolution of reproductive proteins in this important agricultural pest.

Abstract 5022: Generation of T regulatory-cell specific RNA aptamers

Abstract Introduction: RNA aptamers are small RNA oligonucleotides that bind to molecular targets in a manner analogous to antibodies. Theoretically, RNA aptamers have an advantage over antibodies in that large numbers of aptamers can be selected, sequenced and synthesized inexpensively in the laboratory. Here, we report a novel strategy for generating human T regulatory cell (Treg)-specific RNA aptamers. Methods: Human Treg-specific RNA aptamers were developed using whole cell systematic evolution of ligands by exponential enrichment (SELEX). Briefly, a diverse library of 2′fluoro-modified RNA sequences (∼1012) were transcribed from DNA templates containing random 20-mer regions flanked by SEL-2 specific constant sequences. Eight rounds of SELEX were conducted using mononuclear cells from a different donor for each round. Cells were separated into Tregs (CD4+CD25high) and non-Treg CD4 cells (CD4+CD25low) by magnetic cell sorting using a CD4+CD25+ human Treg isolation kit. The aptamer library was pre-cleared using non-Treg CD4 cells. Unbound aptamers were incubated with Tregs from the same donor. Tregs were washed, lysed and Treg-associated aptamers amplified by RT-PCR with SEL2 primers, transcribed and prepared for the next round. Aptamers that bound to human Tregs were collected after each round and sequenced using Illumina-based high-throughput sequencing. Results SELEX was used to successfully enrich for Treg-specific RNA aptamers. Between 2,800,000 and 3,900,000 aptamers were sequenced per round. The number of unique reads decreased from 3,391,279 (initial library) to 594,206 (round 8). At the end of SELEX, 36 unique sequences were found in >1000 copies per million reads. The prevalence of most of these aptamers went up with each round of SELEX. For example, the aptamer designated as Tr-6 was not detected in the unselected pool of aptamers, but 220 copies were found after round 2, 1631 after round 4, and 4832 after round 8. After all 8 rounds, the top 36 sequences represented 25% of the total aptamer population. Twenty-four aptamers were selected for further evaluation based on copy number, consistent enrichment after each round (indicating aptamers that bound to Tregs from each individual donor), bioinformatic properties that predict an aptamer with effective binding, and separation into different sequence and structure families, from which representative sequences were selected. Four aptamers have been tested for their ability to bind to Treg and CD4+ non-Tregs. All bind more extensively to Treg than to CD4+ non-Tregs. Conclusion and future directions: A large number of human Treg-specific RNA aptamers was selected using whole cell SELEX. Ongoing studies are assessing the antigen specificity of the selected aptamers and whether they cross-react with Tregs from other species. The long term goal is to use these aptamers as in vitro diagnostic agents and therapeutically to reduce Treg activity, thereby enhancing the anti-tumor immune response. Citation Format: Suresh Veeramani, Sue E. Blackwell, William H. Thiel, Paloma H. Giangrande, George J. Weiner. Generation of T regulatory-cell specific RNA aptamers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5022. doi:10.1158/1538-7445.AM2015-5022

Understanding the bacterial communities of hard cheese with blowing defect

The environment of hard cheese encourages bacterial synergies and competitions along the ripening process, which might lead in defects such as clostridial blowing. In this study, Denaturing Gradient Gel Electrophoresis (DGGE), a quantitative Clostridium tyrobutyricum PCR and next-generation Illumina-based sequencing of 16S rRNA gene were applied to study 83 Grana Padano spoiled samples. The aim was to investigate the community of clostridia involved in spoilage, the ecological relationships with the other members of the cheese microbiota, and the effect of lysozyme. Three main genera were dominant in the analysed cheeses, Lactobacillus, Streptococcus and Clostridium, and the assignment at the species level was of 94.3% of 4,477,326 high quality sequences. C. tyrobutyricum and C. butyricum were the most prevalent clostridia. Hierarchical clustering based on the abundance of bacterial genera, revealed three main clusters: one characterized by the highest proportion of Clostridium, a second where Lactobacillus was predominant and the last, dominated by Streptococcus thermophilus. Ecological relationships among species were found: cheeses characterized by an high abundance of S. thermophilus and L. rhamnosus were spoiled by C. tyrobutyricum while, when L. delbrueckii was the most abundant Lactobacillus, C. butyricum was the dominant spoiling species. Lysozyme also shaped the bacterial community, reducing C. tyrobutyricum in favour of C. butyricum. Moreover, this preservative increased the proportion of L. delbrueckii and obligate heterofermentative lactobacilli and lowered L. helveticus and non-starter species, such as L. rhamnosus and L. casei.

The immunotranscriptome of the Caribbean reef-building coral Pseudodiploria strigosa.

The viability of coral reefs worldwide has been seriously compromised in the last few decades due in part to the emergence of coral diseases of infectious nature. Despite important efforts to understand the etiology and the contribution of environmental factors associated to coral diseases, the mechanisms of immune response in corals are just beginning to be studied systematically. In this study, we analyzed the set of conserved immune response genes of the Caribbean reef-building coral Pseudodiploria strigosa by Illumina-based transcriptome sequencing and annotation of healthy colonies challenged with whole live Gram-positive and Gram-negative bacteria. Searching the annotated transcriptome with immune-related terms yielded a total of 2782 transcripts predicted to encode conserved immune-related proteins that were classified into three modules: (a) the immune recognition module, containing a wide diversity of putative pattern recognition receptors including leucine-rich repeat-containing proteins, immunoglobulin superfamily receptors, representatives of various lectin families, and scavenger receptors; (b) the intracellular signaling module, containing components from the Toll-like receptor, transforming growth factor, MAPK, and apoptosis signaling pathways; and (3) the effector module, including the C3 and factor B complement components, a variety of proteases and protease inhibitors, and the melanization-inducing phenoloxidase. P. strigosa displays a highly variable and diverse immune recognition repertoire that has likely contributed to its resilience to coral diseases.

Identification of differentially expressed microRNA in the stems and leaves during sugar accumulation in sweet sorghum

MicroRNAs (miRNAs) have been shown to play important roles in plant development, growth and stress response. Sweet sorghum [Sorghum bicolor (L.) Moench] is an important source of bioenergy due to the high sugar content in its stems. However, it is not clear how the miRNA is involved in sugar accumulation in sorghum stems. In order to identify the miRNAs in the stems and the leaves of sweet sorghum, we extracted RNAs of the stems and leaves of sweet sorghum (Rio) and grain sorghum (BTx623) at the heading and dough stages for high-throughput sequencing. A total of 179279048 reads were obtained from Illumina-based sequencing. Further analysis identified nine known miRNAs and twelve novel miRNAs that showed significantly and specifically differentially expressed in the stems of sweet sorghum. The target genes of the differentially expressed novel miRNAs include the transcription factor, glucosyltransferase, protein kinase, cytochrome P450, transporters etc. GO enrichment analysis showed that the predicted targets of these differentially expressed miRNAs participated in diverse physiological and metabolic processes. We performed RT-qRCR analysis on these miRNAs across eight different libraries to validate the miRNAs. Finally, we screened stem-specifically expressed novel miRNA and a leaf-specifically expressed novel miRNA in sweet sorghum comparing with grain sorghum. Our results provide a basis for further investigation of the potential role of these individual miRNAs in sugar accumulation.

Monitoring the dynamics of syntrophic β-oxidizing bacteria during anaerobic degradation of oleic acid by quantitative PCR.

The ecophysiology of long-chain fatty acid-degrading syntrophic β-oxidizing bacteria has been poorly understood due to a lack of quantitative abundance data. Here, TaqMan quantitative PCR (qPCR) assays targeting the 16S rRNA gene of the known mesophilic syntrophic β-oxidizing bacterial genera Syntrophomonas and Syntrophus were developed and validated. Microbial community dynamics were followed using qPCR and Illumina-based high-throughput amplicon sequencing in triplicate methanogenic bioreactors subjected to five consecutive batch feedings of oleic acid. With repeated oleic acid feeding, the initial specific methane production rate significantly increased along with the relative abundances of Syntrophomonas and methanogenic archaea in the bioreactor communities. The novel qPCR assays showed that Syntrophomonas increased from 7 to 31% of the bacterial community 16S rRNA gene concentration, whereas that of Syntrophus decreased from 0.02 to less than 0.005%. High-throughput amplicon sequencing also revealed that Syntrophomonas became the dominant genus within the bioreactor microbiomes. These results suggest that increased specific mineralization rates of oleic acid were attributed to quantitative shifts within the microbial communities toward higher abundances of syntrophic β-oxidizing bacteria and methanogenic archaea. The novel qPCR assays targeting syntrophic β-oxidizing bacteria may thus serve as monitoring tools to indicate the fatty acid β-oxidization potential of anaerobic digester communities.

Metatranscriptomic analyses of honey bee colonies.

Honey bees face numerous biotic threats from viruses to bacteria, fungi, protists, and mites. Here we describe a thorough analysis of microbes harbored by worker honey bees collected from field colonies in geographically distinct regions of Turkey. Turkey is one of the World's most important centers of apiculture, harboring five subspecies of Apis mellifera L., approximately 20% of the honey bee subspecies in the world. We use deep ILLUMINA-based RNA sequencing to capture RNA species for the honey bee and a sampling of all non-endogenous species carried by bees. After trimming and mapping these reads to the honey bee genome, approximately 10% of the sequences (9–10 million reads per library) remained. These were then mapped to a curated set of public sequences containing ca. Sixty megabase-pairs of sequence representing known microbial species associated with honey bees. Levels of key honey bee pathogens were confirmed using quantitative PCR screens. We contrast microbial matches across different sites in Turkey, showing new country recordings of Lake Sinai virus, two Spiroplasma bacterium species, symbionts Candidatus Schmidhempelia bombi, Frischella perrara, Snodgrassella alvi, Gilliamella apicola, Lactobacillus spp.), neogregarines, and a trypanosome species. By using metagenomic analysis, this study also reveals deep molecular evidence for the presence of bacterial pathogens (Melissococcus plutonius, Paenibacillus larvae), Varroa destructor-1 virus, Sacbrood virus, and fungi. Despite this effort we did not detect KBV, SBPV, Tobacco ringspot virus, VdMLV (Varroa Macula like virus), Acarapis spp., Tropilaeleps spp. and Apocephalus (phorid fly). We discuss possible impacts of management practices and honey bee subspecies on microbial retinues. The described workflow and curated microbial database will be generally useful for microbial surveys of healthy and declining honey bees.

Whole-genome sequencing reveals that mutations in myosin-5 confer resistance to the fungicide phenamacril in Fusarium graminearum.

To determine the mechanism of resistance to the fungicide phenamacril (JS399-19) in Fusarium graminearum, the causal agent of Fusarium head blight, we sequenced and annotated the genome of the resistant strain YP-1 (generated by treating the F. graminearum reference strain PH-1 with phenamacril). Of 1.4 million total reads from an Illumina-based paired-end sequencing assay, 92.80% were aligned to the F. graminearum reference genome. Compared with strain PH-1, strain YP-1 contained 1,989 single-nucleotide polymorphisms that led to amino acid mutations in 132 genes. We sequenced 22 functional annotated genes of another F. graminearum sensitive strain (strain 2021) and corresponding resistant strains. The only mutation common to all of the resistant mutants occurred in the gene encoding myosin-5 (point mutations at codon 216, 217, 418, 420, or 786). To confirm whether the mutations in myosin-5 confer resistance to phenamacril, we exchanged the myosin-5 locus between the sensitive strain 2021 and the resistant strain Y2021A by homologous double exchange. The transformed mutants with a copy of the resistant fragment exhibited resistance to phenamacril, and the transformed mutant with a copy of the sensitive fragment exhibited sensitivity to phenamacril. These results indicate that mutations in myosin-5 confers resistance to phenamacril in F. graminearum.

Reemergence and amplification of tuberculosis in the Canadian arctic.

Between November 2011 and November 2012, a Canadian village of 933 persons had 50 culture-positive cases of tuberculosis, with 49 sharing the same genotype. We performed Illumina-based whole-genome sequencing on Mycobacterium tuberculosis isolates from this village, during and before the outbreak. Phylogenetic trees were generated using the maximum likelihood method. Three distinct genotypes were identified. Strain I (n = 7) was isolated in 1991-1996. Strain II (n = 8) was isolated in 1996-2004. Strain III (n = 62) first appeared in 2007 and did not arise from strain I or II. Within strain III, there were 3 related but distinct clusters: IIIA, IIIB, and IIIC. Between 2007 and 2010, cluster IIIA predominated (11 of 22 vs 2 of 40; P < .001), whereas in 2011-2012 clusters IIIB (n = 18) and IIIC (n = 20) predominated over cluster IIIA (n = 11). Combined evolutionary and epidemiologic analysis of strain III cases revealed that the outbreak in 2011-2012 was the result of ≥6 temporally staggered events, spanning from 1 reactivation case to a point-source outbreak of 20 cases. After the disappearance of 2 strains of M. tuberculosis in this village, its reemergence in 2007 was followed by an epidemiologic amplification, affecting >5% of the population.