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September 22, 2019

Single-cell mRNA isoform diversity in the mouse brain.

Alternative mRNA isoform usage is an important source of protein diversity in mammalian cells. This phenomenon has been extensively studied in bulk tissues, however, it remains unclear how this diversity is reflected in single cells.Here we use long-read sequencing technology combined with unique molecular identifiers (UMIs) to reveal patterns of alternative full-length isoform expression in single cells from the mouse brain. We found a surprising amount of isoform diversity, even after applying a conservative definition of what constitutes an isoform. Genes tend to have one or a few isoforms highly expressed and a larger number of isoforms expressed at a low level. However, for many genes, nearly every sequenced mRNA molecule was unique, and many events affected coding regions suggesting previously unknown protein diversity in single cells. Exon junctions in coding regions were less prone to splicing errors than those in non-coding regions, indicating purifying selection on splice donor and acceptor efficiency.Our findings indicate that mRNA isoform diversity is an important source of biological variability also in single cells.


September 22, 2019

Major histocompatibility complex haplotyping and long-amplicon allele discovery in cynomolgus macaques from Chinese breeding facilities.

Very little is currently known about the major histocompatibility complex (MHC) region of cynomolgus macaques (Macaca fascicularis; Mafa) from Chinese breeding centers. We performed comprehensive MHC class I haplotype analysis of 100 cynomolgus macaques from two different centers, with animals from different reported original geographic origins (Vietnamese, Cambodian, and Cambodian/Indonesian mixed-origin). Many of the samples were of known relation to each other (sire, dam, and progeny sets), making it possible to characterize lineage-level haplotypes in these animals. We identified 52 Mafa-A and 74 Mafa-B haplotypes in this cohort, many of which were restricted to specific sample origins. We also characterized full-length MHC class I transcripts using Pacific Biosciences (PacBio) RS II single-molecule real-time (SMRT) sequencing. This technology allows for complete read-through of unfragmented MHC class I transcripts (~1100 bp in length), so no assembly is required to unambiguously resolve novel full-length sequences. Overall, we identified 311 total full-length transcripts in a subset of 72 cynomolgus macaques from these Chinese breeding facilities; 130 of these sequences were novel and an additional 115 extended existing short database sequences to span the complete open reading frame. This significantly expands the number of Mafa-A, Mafa-B, and Mafa-I full-length alleles in the official cynomolgus macaque MHC class I database. The PacBio technique described here represents a general method for full-length allele discovery and genotyping that can be extended to other complex immune loci such as MHC class II, killer immunoglobulin-like receptors, and Fc gamma receptors.


September 22, 2019

The first whole transcriptomic exploration of pre-oviposited early chicken embryos using single and bulked embryonic RNA-sequencing.

The chicken is a valuable model organism, especially in evolutionary and embryology research because its embryonic development occurs in the egg. However, despite its scientific importance, no transcriptome data have been generated for deciphering the early developmental stages of the chicken because of practical and technical constraints in accessing pre-oviposited embryos.Here, we determine the entire transcriptome of pre-oviposited avian embryos, including oocyte, zygote, and intrauterine embryos from Eyal-giladi and Kochav stage I (EGK.I) to EGK.X collected using a noninvasive approach for the first time. We also compare RNA-sequencing data obtained using a bulked embryo sequencing and single embryo/cell sequencing technique. The raw sequencing data were preprocessed with two genome builds, Galgal4 and Galgal5, and the expression of 17,108 and 26,102 genes was quantified in the respective builds. There were some differences between the two techniques, as well as between the two genome builds, and these were affected by the emergence of long intergenic noncoding RNA annotations.The first transcriptome datasets of pre-oviposited early chicken embryos based on bulked and single embryo sequencing techniques will serve as a valuable resource for investigating early avian embryogenesis, for comparative studies among vertebrates, and for novel gene annotation in the chicken genome.


September 22, 2019

Interpreting microbial biosynthesis in the genomic age: Biological and practical considerations.

Genome mining has become an increasingly powerful, scalable, and economically accessible tool for the study of natural product biosynthesis and drug discovery. However, there remain important biological and practical problems that can complicate or obscure biosynthetic analysis in genomic and metagenomic sequencing projects. Here, we focus on limitations of available technology as well as computational and experimental strategies to overcome them. We review the unique challenges and approaches in the study of symbiotic and uncultured systems, as well as those associated with biosynthetic gene cluster (BGC) assembly and product prediction. Finally, to explore sequencing parameters that affect the recovery and contiguity of large and repetitive BGCs assembled de novo, we simulate Illumina and PacBio sequencing of the Salinispora tropica genome focusing on assembly of the salinilactam (slm) BGC.


September 22, 2019

Quantitative metaproteomics highlight the metabolic contributions of uncultured phylotypes in a thermophilic anaerobic digester.

In this study, we used multiple meta-omic approaches to characterize the microbial community and the active metabolic pathways of a stable industrial biogas reactor with food waste as the dominant feedstock, operating at thermophilic temperatures (60°C) and elevated levels of free ammonia (367 mg/liter NH3-N). The microbial community was strongly dominated (76% of all 16S rRNA amplicon sequences) by populations closely related to the proteolytic bacterium Coprothermobacter proteolyticus. Multiple Coprothermobacter-affiliated strains were detected, introducing an additional level of complexity seldom explored in biogas studies. Genome reconstructions provided metabolic insight into the microbes that performed biomass deconstruction and fermentation, including the deeply branching phyla Dictyoglomi and Planctomycetes and the candidate phylum “Atribacteria” These biomass degraders were complemented by a synergistic network of microorganisms that convert key fermentation intermediates (fatty acids) via syntrophic interactions with hydrogenotrophic methanogens to ultimately produce methane. Interpretation of the proteomics data also suggested activity of a Methanosaeta phylotype acclimatized to high ammonia levels. In particular, we report multiple novel phylotypes proposed as syntrophic acetate oxidizers, which also exert expression of enzymes needed for both the Wood-Ljungdahl pathway and ß-oxidation of fatty acids to acetyl coenzyme A. Such an arrangement differs from known syntrophic oxidizing bacteria and presents an interesting hypothesis for future studies. Collectively, these findings provide increased insight into active metabolic roles of uncultured phylotypes and presents new synergistic relationships, both of which may contribute to the stability of the biogas reactor.Biogas production through anaerobic digestion of organic waste provides an attractive source of renewable energy and a sustainable waste management strategy. A comprehensive understanding of the microbial community that drives anaerobic digesters is essential to ensure stable and efficient energy production. Here, we characterize the intricate microbial networks and metabolic pathways in a thermophilic biogas reactor. We discuss the impact of frequently encountered microbial populations as well as the metabolism of newly discovered novel phylotypes that seem to play distinct roles within key microbial stages of anaerobic digestion in this stable high-temperature system. In particular, we draft a metabolic scenario whereby multiple uncultured syntrophic acetate-oxidizing bacteria are capable of syntrophically oxidizing acetate as well as longer-chain fatty acids (via the ß-oxidation and Wood-Ljundahl pathways) to hydrogen and carbon dioxide, which methanogens subsequently convert to methane. Copyright © 2016 American Society for Microbiology.


September 22, 2019

A genomic case study of mixed fibrolamellar hepatocellular carcinoma.

Mixed fibrolamellar hepatocellular carcinoma (mFL-HCC) is a rare liver tumor defined by the presence of both pure FL-HCC and conventional HCC components, represents up to 25% of cases of FL-HCC, and has been associated with worse prognosis. Recent genomic characterization of pure FL-HCC identified a highly recurrent transcript fusion (DNAJB1:PRKACA) not found in conventional HCC.We performed exome and transcriptome sequencing of a case of mFL-HCC. A novel BAC-capture approach was developed to identify a 400 kb deletion as the underlying genomic mechanism for a DNAJB1:PRKACA fusion in this case. A sensitive Nanostring Elements assay was used to screen for this transcript fusion in a second case of mFL-HCC, 112 additional HCC samples and 44 adjacent non-tumor liver samples.We report the first comprehensive genomic analysis of a case of mFL-HCC. No common HCC-associated mutations were identified. The very low mutation rate of this case, large number of mostly single-copy, long-range copy number variants, and high expression of ERBB2 were more consistent with previous reports of pure FL-HCC than conventional HCC. In particular, the DNAJB1:PRKACA fusion transcript specifically associated with pure FL-HCC was detected at very high expression levels. Subsequent analysis revealed the presence of this fusion in all primary and metastatic samples, including those with mixed or conventional HCC pathology. A second case of mFL-HCC confirmed our finding that the fusion was detectable in conventional components. An expanded screen identified a third case of fusion-positive HCC, which upon review, also had both conventional and fibrolamellar features. This screen confirmed the absence of the fusion in all conventional HCC and adjacent non-tumor liver samples.These results indicate that mFL-HCC is similar to pure FL-HCC at the genomic level and the DNAJB1:PRKACA fusion can be used as a diagnostic tool for both pure and mFL-HCC.© The Author 2016. Published by Oxford University Press on behalf of the European Society for Medical Oncology.


September 22, 2019

PCR and omics based techniques to study the diversity, ecology and biology of anaerobic fungi: Insights, challenges andopportunities.

Anaerobic fungi (phylum Neocallimastigomycota) are common inhabitants of the digestive tract of mammalian herbivores, and in the rumen, can account for up to 20% of the microbial biomass. Anaerobic fungi play a primary role in the degradation of lignocellulosic plant material. They also have a syntrophic interaction with methanogenic archaea, which increases their fiber degradation activity. To date, nine anaerobic fungal genera have been described, with further novel taxonomic groupings known to exist based on culture-independent molecular surveys. However, the true extent of their diversity may be even more extensively underestimated as anaerobic fungi continue being discovered in yet unexplored gut and non-gut environments. Additionally many studies are now known to have used primers that provide incomplete coverage of the Neocallimastigomycota. For ecological studies the internal transcribed spacer 1 region (ITS1) has been the taxonomic marker of choice, but due to various limitations the large subunit rRNA (LSU) is now being increasingly used. How the continued expansion of our knowledge regarding anaerobic fungal diversity will impact on our understanding of their biology and ecological role remains unclear; particularly as it is becoming apparent that anaerobic fungi display niche differentiation. As a consequence, there is a need to move beyond the broad generalization of anaerobic fungi as fiber-degraders, and explore the fundamental differences that underpin their ability to exist in distinct ecological niches. Application of genomics, transcriptomics, proteomics and metabolomics to their study in pure/mixed cultures and environmental samples will be invaluable in this process. To date the genomes and transcriptomes of several characterized anaerobic fungal isolates have been successfully generated. In contrast, the application of proteomics and metabolomics to anaerobic fungal analysis is still in its infancy. A central problem for all analyses, however, is the limited functional annotation of anaerobic fungal sequence data. There is therefore an urgent need to expand information held within publicly available reference databases. Once this challenge is overcome, along with improved sample collection and extraction, the application of these techniques will be key in furthering our understanding of the ecological role and impact of anaerobic fungi in the wide range of environments they inhabit.


September 22, 2019

Single-cell isoform RNA sequencing characterizes isoforms in thousands of cerebellar cells.

Full-length RNA sequencing (RNA-Seq) has been applied to bulk tissue, cell lines and sorted cells to characterize transcriptomes, but applying this technology to single cells has proven to be difficult, with less than ten single-cell transcriptomes having been analyzed thus far. Although single splicing events have been described for =200 single cells with statistical confidence, full-length mRNA analyses for hundreds of cells have not been reported. Single-cell short-read 3′ sequencing enables the identification of cellular subtypes, but full-length mRNA isoforms for these cell types cannot be profiled. We developed a method that starts with bulk tissue and identifies single-cell types and their full-length RNA isoforms without fluorescence-activated cell sorting. Using single-cell isoform RNA-Seq (ScISOr-Seq), we identified RNA isoforms in neurons, astrocytes, microglia, and cell subtypes such as Purkinje and Granule cells, and cell-type-specific combination patterns of distant splice sites. We used ScISOr-Seq to improve genome annotation in mouse Gencode version 10 by determining the cell-type-specific expression of 18,173 known and 16,872 novel isoforms.


September 22, 2019

Multi-platform assessment of transcriptome profiling using RNA-seq in the ABRF next-generation sequencing study.

High-throughput RNA sequencing (RNA-seq) greatly expands the potential for genomics discoveries, but the wide variety of platforms, protocols and performance capabilitites has created the need for comprehensive reference data. Here we describe the Association of Biomolecular Resource Facilities next-generation sequencing (ABRF-NGS) study on RNA-seq. We carried out replicate experiments across 15 laboratory sites using reference RNA standards to test four protocols (poly-A-selected, ribo-depleted, size-selected and degraded) on five sequencing platforms (Illumina HiSeq, Life Technologies PGM and Proton, Pacific Biosciences RS and Roche 454). The results show high intraplatform (Spearman rank R > 0.86) and inter-platform (R > 0.83) concordance for expression measures across the deep-count platforms, but highly variable efficiency and cost for splice junction and variant detection between all platforms. For intact RNA, gene expression profiles from rRNA-depletion and poly-A enrichment are similar. In addition, rRNA depletion enables effective analysis of degraded RNA samples. This study provides a broad foundation for cross-platform standardization, evaluation and improvement of RNA-seq.


September 22, 2019

PacBio sequencing and its applications.

Single-molecule, real-time sequencing developed by Pacific BioSciences offers longer read lengths than the second-generation sequencing (SGS) technologies, making it well-suited for unsolved problems in genome, transcriptome, and epigenetics research. The highly-contiguous de novo assemblies using PacBio sequencing can close gaps in current reference assemblies and characterize structural variation (SV) in personal genomes. With longer reads, we can sequence through extended repetitive regions and detect mutations, many of which are associated with diseases. Moreover, PacBio transcriptome sequencing is advantageous for the identification of gene isoforms and facilitates reliable discoveries of novel genes and novel isoforms of annotated genes, due to its ability to sequence full-length transcripts or fragments with significant lengths. Additionally, PacBio’s sequencing technique provides information that is useful for the direct detection of base modifications, such as methylation. In addition to using PacBio sequencing alone, many hybrid sequencing strategies have been developed to make use of more accurate short reads in conjunction with PacBio long reads. In general, hybrid sequencing strategies are more affordable and scalable especially for small-size laboratories than using PacBio Sequencing alone. The advent of PacBio sequencing has made available much information that could not be obtained via SGS alone. Copyright © 2015 The Authors. Production and hosting by Elsevier Ltd.. All rights reserved.


September 22, 2019

Analyses of alternative polyadenylation: from old school biochemistry to high-throughput technologies.

Alternations in usage of polyadenylation sites during transcription termination yield transcript isoforms from a gene. Recent findings of transcriptome-wide alternative polyadenylation (APA) as a molecular response to changes in biology position APA not only as a molecular event of early transcriptional termination but also as a cellular regulatory step affecting various biological pathways. With the development of high-throughput profiling technologies at a single nucleotide level and their applications targeted to the 3′-end of mRNAs, dynamics in the landscape of mRNA 3′-end is measureable at a global scale. In this review, methods and technologies that have been adopted to study APA events are discussed. In addition, various bioinformatics algorithms for APA isoform analysis using publicly available RNA-seq datasets are introduced. [BMB Reports 2017; 50(4): 201-207].


September 22, 2019

The full transcription map of mouse papillomavirus type 1 (MmuPV1) in mouse wart tissues.

Mouse papillomavirus type 1 (MmuPV1) provides, for the first time, the opportunity to study infection and pathogenesis of papillomaviruses in the context of laboratory mice. In this report, we define the transcriptome of MmuPV1 genome present in papillomas arising in experimentally infected mice using a combination of RNA-seq, PacBio Iso-seq, 5′ RACE, 3′ RACE, primer-walking RT-PCR, RNase protection, Northern blot and in situ hybridization analyses. We demonstrate that the MmuPV1 genome is transcribed unidirectionally from five major promoters (P) or transcription start sites (TSS) and polyadenylates its transcripts at two major polyadenylation (pA) sites. We designate the P7503, P360 and P859 as “early” promoters because they give rise to transcripts mostly utilizing the polyadenylation signal at nt 3844 and therefore can only encode early genes, and P7107 and P533 as “late” promoters because they give rise to transcripts utilizing polyadenylation signals at either nt 3844 or nt 7047, the latter being able to encode late, capsid proteins. MmuPV1 genome contains five splice donor sites and three acceptor sites that produce thirty-six RNA isoforms deduced to express seven predicted early gene products (E6, E7, E1, E1^M1, E1^M2, E2 and E8^E2) and three predicted late gene products (E1^E4, L2 and L1). The majority of the viral early transcripts are spliced once from nt 757 to 3139, while viral late transcripts, which are predicted to encode L1, are spliced twice, first from nt 7243 to either nt 3139 (P7107) or nt 757 to 3139 (P533) and second from nt 3431 to nt 5372. Thirteen of these viral transcripts were detectable by Northern blot analysis, with the P533-derived late E1^E4 transcripts being the most abundant. The late transcripts could be detected in highly differentiated keratinocytes of MmuPV1-infected tissues as early as ten days after MmuPV1 inoculation and correlated with detection of L1 protein and viral DNA amplification. In mature warts, detection of L1 was also found in more poorly differentiated cells, as previously reported. Subclinical infections were also observed. The comprehensive transcription map of MmuPV1 generated in this study provides further evidence that MmuPV1 is similar to high-risk cutaneous beta human papillomaviruses. The knowledge revealed will facilitate the use of MmuPV1 as an animal virus model for understanding of human papillomavirus gene expression, pathogenesis and immunology.


September 22, 2019

PacBio for haplotyping in gene families.

The throughput and read length provided by Pacific Bioscience (PacBio) Single Molecule Real Time (SMRT) sequencing platform makes it feasible to construct contiguous, non-chimeric sequences. This is especially useful for genes with repetitive sequences in their gene bodies in gene families. We illustrate the use of PacBio to sequence and assemble hundreds of transcripts of gluten gene families from different cultivars of wheat using sequence from a single SMRT cell. To this end, we barcoded amplicons from different cultivars, then pooled these into one library for sequencing. Sequencing reads were later separated by the barcodes and further sorted into different gene groups by blast. The reads from each gene are then assembled by SeqmanNGen software. Given the length of 1 kb for each sequence derived from an initial molecule, the phase of the polymorphisms is not lost and can be used to infer also haplotype differences between different cultivars.


September 22, 2019

Single-cell multiomics: multiple measurements from single cells.

Single-cell sequencing provides information that is not confounded by genotypic or phenotypic heterogeneity of bulk samples. Sequencing of one molecular type (RNA, methylated DNA or open chromatin) in a single cell, furthermore, provides insights into the cell’s phenotype and links to its genotype. Nevertheless, only by taking measurements of these phenotypes and genotypes from the same single cells can such inferences be made unambiguously. In this review, we survey the first experimental approaches that assay, in parallel, multiple molecular types from the same single cell, before considering the challenges and opportunities afforded by these and future technologies. Copyright © 2016. Published by Elsevier Ltd.


September 22, 2019

Root endophytes and invasiveness: no difference between native and non-native Phragmites in the Great Lakes Region

Microbial interactions could play an important role in plant invasions. If invasive plants associate with relatively more mutualists or fewer pathogens than their native counterparts, then microbial communities could foster plant invasiveness. Studies examining the effects of microbes on invasive plants commonly focus on a single microbial group (e.g., bacteria) or measure only plant response to microbes, not documenting the specific taxa associating with invaders. We surveyed root microbial communities associated with co-occurring native and non-native lineages of Phragmites australis, across Michigan, USA. Our aim was to determine whether (1) plant lineage was a stronger predictor of root microbial community composition than environmental variables and (2) the non-native lineage associated with more mutualistic and/or fewer pathogenic microbes than the native lineage. We used microscopy and culture-independent molecular methods to examine fungal colonization rate and community composition in three major microbial groups (bacteria, fungi, and oomycetes) within roots. We also used microbial functional databases to assess putative functions of the observed microbial taxa. While fungal colonization of roots was significantly higher in non-native Phragmites than the native lineage, we found no differences in root microbial community composition or potential function between the two Phragmites lineages. Community composition did differ significantly by site, with soil saturation playing a significant role in structuring communities in all three microbial groups. The relative abundance of some specific bacterial taxa did differ between Phragmites lineages at the phylum and genus level (e.g., Proteobacteria, Firmicutes). Purported function of root fungi and respiratory mode of root bacteria also did not differ between native and non-native Phragmites. We found no evidence that native and non-native Phragmites harbored distinct root microbial communities; nor did those communities differ functionally. Therefore, if the trends revealed at our sites are widespread, it is unlikely that total root microbial communities are driving invasion by non-native Phragmites plants.


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