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Asset Tag: Circular consensus sequencing

September 22, 2019

Sequencing 16S rRNA gene fragments using the PacBio SMRT DNA sequencing system.

Over the past 10 years, microbial ecologists have largely abandoned sequencing 16S rRNA genes by the Sanger sequencing method and have instead adopted highly parallelized sequencing platforms. These new platforms, such as 454 and Illumina’s MiSeq, have allowed researchers to obtain millions of high quality but short sequences. The result of the added sequencing depth has been significant improvements in experimental design. The tradeoff has been the decline in the number of full-length reference sequences that are deposited into databases. To overcome this problem, we tested the ability of the PacBio Single Molecule, Real-Time (SMRT) DNA sequencing platform to generate sequence reads from the 16S rRNA gene. We generated sequencing data from the V4, V3-V5, V1-V3, V1-V5, V1-V6, and V1-V9 variable regions from within the 16S rRNA gene using DNA from a synthetic mock community and natural samples collected from human feces, mouse feces, and soil. The mock community allowed us to assess the actual sequencing error rate and how that error rate changed when different curation methods were applied. We developed a simple method based on sequence characteristics and quality scores to reduce the observed error rate for the V1-V9 region from 0.69 to 0.027%. This error rate is comparable to what has been observed for the shorter reads generated by 454 and Illumina’s MiSeq sequencing platforms. Although the per base sequencing cost is still significantly more than that of MiSeq, the prospect of supplementing reference databases with full-length sequences from organisms below the limit of detection from the Sanger approach is exciting.

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September 22, 2019

Initial colonization, community assembly and ecosystem function: fungal colonist traits and litter biochemistry mediate decay rate.

Priority effects are an important ecological force shaping biotic communities and ecosystem processes, in which the establishment of early colonists alters the colonization success of later-arriving organisms via competitive exclusion and habitat modification. However, we do not understand which biotic and abiotic conditions lead to strong priority effects and lasting historical contingencies. Using saprotrophic fungi in a model leaf decomposition system, we investigated whether compositional and functional consequences of initial colonization were dependent on initial colonizer traits, resource availability or a combination thereof. To test these ideas, we factorially manipulated leaf litter biochemistry and initial fungal colonist identity, quantifying subsequent community composition, using neutral genetic markers, and community functional characteristics, including enzyme potential and leaf decay rates. During the first 3 months, initial colonist respiration rate and physiological capacity to degrade plant detritus were significant determinants of fungal community composition and leaf decay, indicating that rapid growth and lignolytic potential of early colonists contributed to altered trajectories of community assembly. Further, initial colonization on oak leaves generated increasingly divergent trajectories of fungal community composition and enzyme potential, indicating stronger initial colonizer effects on energy-poor substrates. Together, these observations provide evidence that initial colonization effects, and subsequent consequences on litter decay, are dependent upon substrate biochemistry and physiological traits within a regional species pool. Because microbial decay of plant detritus is important to global C storage, our results demonstrate that understanding the mechanisms by which initial conditions alter priority effects during community assembly may be key to understanding the drivers of ecosystem-level processes. © 2015 John Wiley & Sons Ltd.

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September 22, 2019

The features of mucosa-associated microbiota in primary sclerosing cholangitis.

Little is known about the role of the microbiome in primary sclerosing cholangitis.To explore the mucosa-associated microbiota in primary sclerosing cholangitis (PSC) patients across different locations in the gut, and to compare it with inflammatory bowel disease (IBD)-only patients and healthy controls.Biopsies from the terminal ileum, right colon, and left colon were collected from patients and healthy controls undergoing colonoscopy. Microbiota profiling using bacterial 16S rRNA sequencing was performed on all biopsies.Forty-four patients were recruited: 20 with PSC (19 with PSC-IBD and one with PSC-only), 15 with IBD-only and nine healthy controls. The overall microbiome profile was similar throughout different locations in the gut. No differences in the global microbiome profile were found. However, we observed significant PSC-associated enrichment in Barnesiellaceae at the family level, and in Blautia and an unidentified Barnesiellaceae at the genus level. At the operational taxa unit level, most shifts in PSC were observed in Clostridiales and Bacteroidales orders, with approximately 86% of shifts occurring within the former order.The overall microbiota profile was similar across multiple locations in the gut from the same individual regardless of disease status. In this study, the mucosa associated-microbiota of patients with primary sclerosing cholangitis was characterised by enrichment of Blautia and Barnesiellaceae and by major shifts in operational taxa units within Clostridiales order.© 2016 John Wiley & Sons Ltd.

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September 22, 2019

Complex effects of mammalian grazing on extramatrical mycelial biomass in the Scandes forest-tundra ecotone.

Mycorrhizal associations are widespread in high-latitude ecosystems and are potentially of great importance for global carbon dynamics. Although large herbivores play a key part in shaping subarctic plant communities, their impact on mycorrhizal dynamics is largely unknown. We measured extramatrical mycelial (EMM) biomass during one growing season in 16-year-old herbivore exclosures and unenclosed control plots (ambient), at three mountain birch forests and two shrub heath sites, in the Scandes forest-tundra ecotone. We also used high-throughput amplicon sequencing for taxonomic identification to investigate differences in fungal species composition. At the birch forest sites, EMM biomass was significantly higher in exclosures (1.36 ± 0.43 g C/m2) than in ambient conditions (0.66 ± 0.17 g C/m2) and was positively influenced by soil thawing degree-days. At the shrub heath sites, there was no significant effect on EMM biomass (exclosures: 0.72 ± 0.09 g C/m2; ambient plots: 1.43 ± 0.94). However, EMM biomass was negatively related toBetula nanaabundance, which was greater in exclosures, suggesting that grazing affected EMM biomass positively. We found no significant treatment effects on fungal diversity but the most abundant ectomycorrhizal lineage/cortinarius, showed a near-significant positive effect of herbivore exclusion (p = .08), indicating that herbivory also affects fungal community composition. These results suggest that herbivory can influence fungal biomass in highly context-dependent ways in subarctic ecosystems. Considering the importance of root-associated fungi for ecosystem carbon balance, these findings could have far-reaching implications.

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September 22, 2019

Transcriptome-referenced association study of clove shape traits in garlic.

Genome-wide association studies are a powerful approach for identifying genes related to complex traits in organisms, but are limited by the requirement for a reference genome sequence of the species under study. To circumvent this problem, we propose a transcriptome-referenced association study (TRAS) that utilizes a transcriptome generated by single-molecule long-read sequencing as a reference sequence to score population variation at both transcript sequence and expression levels. Candidate transcripts are identified when both scores are associated with a trait and their potential interactions are ascertained by expression quantitative trait loci analysis. Applying this method to characterize garlic clove shape traits in 102 landraces, we identified 22 candidate transcripts, most of which showed extensive interactions. Eight transcripts were long non-coding RNAs (lncRNAs), and the others were proteins involved mainly in carbohydrate metabolism, protein degradation, etc. TRAS, as an efficient tool for association study independent of a reference genome, extends the applicability of association studies to a broad range of species.

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September 22, 2019

Identification of putative coffee rust mycoparasites using single molecule DNA sequencing of infected pustules.

The interaction of crop pests with their natural enemies is a fundament to their control. Natural enemies of fungal pathogens of crops are poorly known relative to those of insect pests despite the diversity of fungal pathogens and their economic importance. Currently, many regions across Latin America are experiencing unprecedented epidemics of coffee rust (Hemileia vastatrix). Identification of natural enemies of coffee rust could aid in developing management strategies or in pinpointing species that could be used for biocontrol. Here we characterize fungal communities associated with coffee rust lesions by single molecule DNA sequencing of fungal ribosomal RNA barcodes from leaf discs (˜28 mm(2)) containing rust lesions and control discs with no rust lesions. The leaf disc communities were hyper-diverse in fungi, with up to 57 taxa per control disc, and the diversity was only slightly reduced in rust-infected discs. However, geography had a greater influence on the fungal community than whether the disk was infected by coffee rust. Through comparisons between control and rust-infected leaf discs, as well as taxonomic criteria, we identified 15 putative mycoparasitic fungi. These fungi are concentrated in fungal family Cordycipitaceae and order Tremellales. These data emphasize the complexity of fungal diversity of unknown ecological function within a leaf that might influence plant disease epidemics or lead to the development of species for biocontrol of fungal disease. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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September 22, 2019

Laboratory colonization stabilizes the naturally dynamic microbiome composition of field collected Dermacentor andersoni ticks.

Nearly a quarter of emerging infectious diseases identified in the last century are arthropod-borne. Although ticks and insects can carry pathogenic microorganisms, non-pathogenic microbes make up the majority of their microbial communities. The majority of tick microbiome research has had a focus on discovery and description; very few studies have analyzed the ecological context and functional responses of the bacterial microbiome of ticks. The goal of this analysis was to characterize the stability of the bacterial microbiome of Dermacentor andersoni ticks between generations and two populations within a species.The bacterial microbiome of D. andersoni midguts and salivary glands was analyzed from populations collected at two different ecologically distinct sites by comparing field (F1) and lab-reared populations (F1-F3) over three generations. The microbiome composition of pooled and individual samples was analyzed by sequencing nearly full-length 16S rRNA gene amplicons using a Pacific Biosciences CCS platform that allows identification of bacteria to the species level.In this study, we found that the D. andersoni microbiome was distinct in different geographic populations and was tissue specific, differing between the midgut and the salivary gland, over multiple generations. Additionally, our study showed that the microbiomes of laboratory-reared populations were not necessarily representative of their respective field populations. Furthermore, we demonstrated that the microbiome of a few individual ticks does not represent the microbiome composition at the population level.We demonstrated that the bacterial microbiome of D. andersoni was complex over three generations and specific to tick tissue (midgut vs. salivary glands) as well as geographic location (Burns, Oregon vs. Lake Como, Montana vs. laboratory setting). These results provide evidence that habitat of the tick population is a vital component of the complexity of the bacterial microbiome of ticks, and that the microbiome of lab colonies may not allow for comparative analyses with field populations. A broader understanding of microbiome variation will be required if we are to employ manipulation of the microbiome as a method for interfering with acquisition and transmission of tick-borne pathogens.

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September 22, 2019

Anthropogenic N deposition alters the composition of expressed class II fungal peroxidases.

Here, we present evidence that ca. 20 years of experimental N deposition altered the composition of lignin-decaying class II peroxidases expressed by forest floor fungi, a response which has occurred concurrently with reductions in plant litter decomposition and a rapid accumulation of soil organic matter. This finding suggests that anthropogenic N deposition has induced changes in the biological mediation of lignin decay, the rate limiting step in plant litter decomposition. Thus, an altered composition of transcripts for a critical gene that is associated with terrestrial C cycling may explain the increased soil C storage under long-term increases in anthropogenic N deposition.IMPORTANCE Fungal class II peroxidases are enzymes that mediate the rate-limiting step in the decomposition of plant material, which involves the oxidation of lignin and other polyphenols. In field experiments, anthropogenic N deposition has increased soil C storage in forests, a result which could potentially arise from anthropogenic N-induced changes in the composition of class II peroxidases expressed by the fungal community. In this study, we have gained unique insight into how anthropogenic N deposition, a widespread agent of global change, affects the expression of a functional gene encoding an enzyme that plays a critical role in a biologically mediated ecosystem process. Copyright © 2018 American Society for Microbiology.

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September 22, 2019

Research benefits of storing genitourinary samples: 16S rRNA sequencing to evaluate vaginal bacterial communities.

Using well-characterised, but old and carefully frozen genital tract research samples may be a cost-effective way of performing metagenomic studies, but risks loss of low abundance (but relevant) bacterial species DNA. Moi et al.1 used 16S rRNA and UreDNA sequencing to detect ureaplasmas in frozen urine samples collected from 362 men with NGU in 2010–2011. They found that urethral inflammatory responses to ureaplasmas were less severe than to Chlamydia trachomatis and Mycoplasma genitalium.

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September 22, 2019

Long-read sequencing of nascent RNA reveals coupling among RNA processing events.

Pre-mRNA splicing is accomplished by the spliceosome, a megadalton complex that assembles de novo on each intron. Because spliceosome assembly and catalysis occur cotranscriptionally, we hypothesized that introns are removed in the order of their transcription in genomes dominated by constitutive splicing. Remarkably little is known about splicing order and the regulatory potential of nascent transcript remodeling by splicing, due to the limitations of existing methods that focus on analysis of mature splicing products (mRNAs) rather than substrates and intermediates. Here, we overcome this obstacle through long-read RNA sequencing of nascent, multi-intron transcripts in the fission yeast Schizosaccharomyces pombe Most multi-intron transcripts were fully spliced, consistent with rapid cotranscriptional splicing. However, an unexpectedly high proportion of transcripts were either fully spliced or fully unspliced, suggesting that splicing of any given intron is dependent on the splicing status of other introns in the transcript. Supporting this, mild inhibition of splicing by a temperature-sensitive mutation in prp2, the homolog of vertebrate U2AF65, increased the frequency of fully unspliced transcripts. Importantly, fully unspliced transcripts displayed transcriptional read-through at the polyA site and were degraded cotranscriptionally by the nuclear exosome. Finally, we show that cellular mRNA levels were reduced in genes with a high number of unspliced nascent transcripts during caffeine treatment, showing regulatory significance of cotranscriptional splicing. Therefore, overall splicing of individual nascent transcripts, 3′ end formation, and mRNA half-life depend on the splicing status of neighboring introns, suggesting crosstalk among spliceosomes and the polyA cleavage machinery during transcription elongation.© 2018 Herzel et al.; Published by Cold Spring Harbor Laboratory Press.

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September 22, 2019

Identification of differentially expressed splice variants by the proteogenomic pipeline Splicify.

Proteogenomics, i.e. comprehensive integration of genomics and proteomics data, is a powerful approach identifying novel protein biomarkers. This is especially the case for proteins that differ structurally between disease and control conditions. As tumor development is associated with aberrant splicing, we focus on this rich source of cancer specific biomarkers. To this end, we developed a proteogenomic pipeline, Splicify, which is able to detect differentially expressed protein isoforms. Splicify is based on integrating RNA massive parallel sequencing data and tandem mass spectrometry proteomics data to identify protein isoforms resulting from differential splicing between two conditions. Proof of concept was obtained by applying Splicify to RNA sequencing and mass spectrometry data obtained from colorectal cancer cell line SW480, before and after siRNA-mediated down-modulation of the splicing factors SF3B1 and SRSF1. These analyses revealed 2172 and 149 differentially expressed isoforms, respectively, with peptide confirmation upon knock-down of SF3B1 and SRSF1 compared to their controls. Splice variants identified included RAC1, OSBPL3, MKI67 and SYK. One additional sample was analyzed by PacBio Iso-Seq full-length transcript sequencing after SF3B1 down-modulation. This analysis verified the alternative splicing identified by Splicify and in addition identified novel splicing events that were not represented in the human reference genome annotation. Therefore, Splicify offers a validated proteogenomic data analysis pipeline for identification of disease specific protein biomarkers resulting from mRNA alternative splicing. Splicify is publicly available on GitHub (https://github.com/NKI-TGO/SPLICIFY) and suitable to address basic research questions using pre-clinical model systems as well as translational research questions using patient-derived samples, e.g. allowing to identify clinically relevant biomarkers. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

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September 22, 2019

No assembly required: Full-length MHC class I allele discovery by PacBio circular consensus sequencing.

Single-molecule real-time (SMRT) sequencing technology with the Pacific Biosciences (PacBio) RS II platform offers the potential to obtain full-length coding regions (~1100-bp) from MHC class I cDNAs. Despite the relatively high error rate associated with SMRT technology, high quality sequences can be obtained by circular consensus sequencing (CCS) due to the random nature of the error profile. In the present study we first validated the ability of SMRT-CCS to accurately identify class I transcripts in Mauritian-origin cynomolgus macaques (Macaca fascicularis) that have been characterized previously by cloning and Sanger-based sequencing as well as pyrosequencing approaches. We then applied this SMRT-CCS method to characterize 60 novel full-length class I transcript sequences expressed by a cohort of cynomolgus macaques from China. The SMRT-CCS method described here provides a straightforward protocol for characterization of unfragmented single-molecule cDNA transcripts that will potentially revolutionize MHC class I allele discovery in nonhuman primates and other species. Published by Elsevier Inc.

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September 22, 2019

Profiling of metabolome and bacterial community dynamics in ensiled Medicago sativa inoculated without or with Lactobacillus plantarum or Lactobacillus buchneri.

Using gas chromatography mass spectrometry and the PacBio single molecule with real-time sequencing technology (SMRT), we analyzed the detailed metabolomic profiles and microbial community dynamics involved in ensiled Medicago sativa (alfalfa) inoculated without or with the homofermenter Lactobacillus plantarum or heterofermenter Lactobacillus buchneri. Our results revealed that 280 substances and 102 different metabolites were present in ensiled alfalfa. Inoculation of L. buchneri led to remarkable up-accumulation in concentrations of 4-aminobutyric acid, some free amino acids, and polyols in ensiled alfalfa, whereas considerable down-accumulation in cadaverine and succinic acid were observed in L. plantarum-inoculated silages. Completely different microbial flora and their successions during ensiling were observed in the control and two types of inoculant-treated silages. Inoculation of the L. plantarum or L. buchneri alters the microbial composition dynamics of the ensiled forage in very different manners. Our study demonstrates that metabolomic profiling analysis provides a deep insight in metabolites in silage. Moreover, the PacBio SMRT method revealed the microbial composition and its succession during the ensiling process at the species level. This provides information regarding the microbial processes underlying silage formation and may contribute to target-based regulation methods to achieve high-quality silage production.

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September 22, 2019

Somatic mosaicism of an intragenic FANCB duplication in both fibroblast and peripheral blood cells observed in a Fanconi anemia patient leads to milder phenotype.

Fanconi anemia (FA) is a rare disorder characterized by congenital malformations, progressive bone marrow failure, and predisposition to cancer. Patients harboring X-linked FANCB pathogenic variants usually present with severe congenital malformations resembling VACTERL syndrome with hydrocephalus.We employed the diepoxybutane (DEB) test for FA diagnosis, arrayCGH for detection of duplication, targeted capture and next-gen sequencing for defining the duplication breakpoint, PacBio sequencing of full-length FANCB aberrant transcript, FANCD2 ubiquitination and foci formation assays for the evaluation of FANCB protein function by viral transduction of FANCB-null cells with lentiviral FANCB WT and mutant expression constructs, and droplet digital PCR for quantitation of the duplication in the genomic DNA and cDNA.We describe here an FA-B patient with a mild phenotype. The DEB diagnostic test for FA revealed somatic mosaicism. We identified a 9154 bp intragenic duplication in FANCB, covering the first coding exon 3 and the flanking regions. A four bp homology (GTAG) present at both ends of the breakpoint is consistent with microhomology-mediated duplication mechanism. The duplicated allele gives rise to an aberrant transcript containing exon 3 duplication, predicted to introduce a stop codon in FANCB protein (p.A319*). Duplication levels in the peripheral blood DNA declined from 93% to 7.9% in the span of eleven years. Moreover, the patient fibroblasts have shown 8% of wild-type (WT) allele and his carrier mother showed higher than expected levels of WT allele (79% vs. 50%) in peripheral blood, suggesting that the duplication was highly unstable.Unlike sequence point variants, intragenic duplications are difficult to precisely define, accurately quantify, and may be very unstable, challenging the proper diagnosis. The reversion of genomic duplication to the WT allele results in somatic mosaicism and may explain the relatively milder phenotype displayed by the FA-B patient described here.© 2017 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.

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September 22, 2019

Single-cell (meta-)genomics of a dimorphic Candidatus Thiomargarita nelsonii reveals genomic plasticity.

The genus Thiomargarita includes the world’s largest bacteria. But as uncultured organisms, their physiology, metabolism, and basis for their gigantism are not well understood. Thus, a genomics approach, applied to a single Candidatus Thiomargarita nelsonii cell was employed to explore the genetic potential of one of these enigmatic giant bacteria. The Thiomargarita cell was obtained from an assemblage of budding Ca. T. nelsonii attached to a provannid gastropod shell from Hydrate Ridge, a methane seep offshore of Oregon, USA. Here we present a manually curated genome of Bud S10 resulting from a hybrid assembly of long Pacific Biosciences and short Illumina sequencing reads. With respect to inorganic carbon fixation and sulfur oxidation pathways, the Ca. T. nelsonii Hydrate Ridge Bud S10 genome was similar to marine sister taxa within the family Beggiatoaceae. However, the Bud S10 genome contains genes suggestive of the genetic potential for lithotrophic growth on arsenite and perhaps hydrogen. The genome also revealed that Bud S10 likely respires nitrate via two pathways: a complete denitrification pathway and a dissimilatory nitrate reduction to ammonia pathway. Both pathways have been predicted, but not previously fully elucidated, in the genomes of other large, vacuolated, sulfur-oxidizing bacteria. Surprisingly, the genome also had a high number of unusual features for a bacterium to include the largest number of metacaspases and introns ever reported in a bacterium. Also present, are a large number of other mobile genetic elements, such as insertion sequence (IS) transposable elements and miniature inverted-repeat transposable elements (MITEs). In some cases, mobile genetic elements disrupted key genes in metabolic pathways. For example, a MITE interrupts hupL, which encodes the large subunit of the hydrogenase in hydrogen oxidation. Moreover, we detected a group I intron in one of the most critical genes in the sulfur oxidation pathway, dsrA. The dsrA group I intron also carried a MITE sequence that, like the hupL MITE family, occurs broadly across the genome. The presence of a high degree of mobile elements in genes central to Thiomargarita’s core metabolism has not been previously reported in free-living bacteria and suggests a highly mutable genome.

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