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September 22, 2019

Analysis of gut microbiota – An ever changing landscape.

In the last two decades, the field of metagenomics has greatly expanded due to improvement in sequencing technologies allowing for a more comprehensive characterization of microbial communities. The use of these technologies has led to an unprecedented understanding of human, animal, and environmental microbiomes and have shown that the gut microbiota are comparable to an organ that is intrinsically linked with a variety of diseases. Characterization of microbial communities using next-generation sequencing-by-synthesis approaches have revealed important shifts in microbiota associated with debilitating diseases such as Clostridium difficile infection. But due to limitations in sequence read length, primer biases, and the quality of databases, genus- and species-level classification have been difficult. Third-generation technologies, such as Pacific Biosciences’ single molecule, real-time (SMRT) approach, allow for unbiased, more specific identification of species that are likely clinically relevant. Comparison of Illumina next-generation characterization and SMRT sequencing of samples from patients treated for C. difficile infection revealed similarities in community composition at the phylum and family levels, but SMRT sequencing further allowed for species-level characterization – permitting a better understanding of the microbial ecology of this disease. Thus, as sequencing technologies continue to advance, new species-level insights can be gained in the study of complex and clinically-relevant microbial communities.

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September 22, 2019

CRISPR/Cas9 deletions in a conserved exon of Distal-less generates gains and losses in a recently acquired morphological novelty in flies.

Distal-less has been repeatedly co-opted for the development of many novel traits. Here, we document its curious role in the development of a novel abdominal appendage (“sternite brushes”) in sepsid flies. CRISPR/Cas9 deletions in the homeodomain result in losses of sternite brushes, demonstrating that Distal-less is necessary for their development. However, deletions in the upstream coding exon (Exon 2) produce losses or gains of brushes. A dissection of Exon 2 reveals that the likely mechanism for gains involves a deletion in an exon-splicing enhancer site that leads to exon skipping. Such contradictory phenotypes are also observed in butterflies, suggesting that mutations in the conserved upstream regions have the potential to generate phenotypic variability in insects that diverged 300 million years ago. Our results demonstrate the importance of Distal-less for the development of a novel abdominal appendage in insects and highlight how site-specific mutations in the same exon can produce contradictory phenotypes. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

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September 22, 2019

Isoform sequencing and state-of-art applications for unravelling complexity of plant transcriptomes

Single-molecule real-time (SMRT) sequencing developed by PacBio, also called third-generation sequencing (TGS), offers longer reads than the second-generation sequencing (SGS). Given its ability to obtain full-length transcripts without assembly, isoform sequencing (Iso-Seq) of transcriptomes by PacBio is advantageous for genome annotation, identification of novel genes and isoforms, as well as the discovery of long non-coding RNA (lncRNA). In addition, Iso-Seq gives access to the direct detection of alternative splicing, alternative polyadenylation (APA), gene fusion, and DNA modifications. Such applications of Iso-Seq facilitate the understanding of gene structure, post-transcriptional regulatory networks, and subsequently proteomic diversity. In this review, we summarize its applications in plant transcriptome study, specifically pointing out challenges associated with each step in the experimental design and highlight the development of bioinformatic pipelines. We aim to provide the community with an integrative overview and a comprehensive guidance to Iso-Seq, and thus to promote its applications in plant research.

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September 22, 2019

Single molecule real-time (SMRT) sequencing comes of age: applications and utilities for medical diagnostics.

Short read massive parallel sequencing has emerged as a standard diagnostic tool in the medical setting. However, short read technologies have inherent limitations such as GC bias, difficulties mapping to repetitive elements, trouble discriminating paralogous sequences, and difficulties in phasing alleles. Long read single molecule sequencers resolve these obstacles. Moreover, they offer higher consensus accuracies and can detect epigenetic modifications from native DNA. The first commercially available long read single molecule platform was the RS system based on PacBio’s single molecule real-time (SMRT) sequencing technology, which has since evolved into their RSII and Sequel systems. Here we capsulize how SMRT sequencing is revolutionizing constitutional, reproductive, cancer, microbial and viral genetic testing.© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.

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September 22, 2019

Dual platform long-read RNA-sequencing dataset of the human Cytomegalovirus Lytic transcriptome

RNA-sequencing has revolutionized transcriptomics and the way we measure gene expression (Wang et al., 2009). As of today, short-read RNA sequencing is more widely used, and due to its low price and high throughput, is the preferred tool for the quantitative analysis of gene expression. However, the annotation of transcript isoforms is rather difficult using only short-read sequencing data, because the reads are shorter than most transcripts (Steijger et al., 2013). Long-read sequencing, on the other hand, can provide full contig information about transcripts, including exon-connectivity, and its merits in transcriptome profiling are being increasingly acknowledged (Sharon et al., 2013; Abdel-Ghany et al., 2016; Wang et al., 2016; Kuo et al., 2017). Due to the relatively low throughput of current long-read sequencing technologies, they can only characterize smaller transcriptomes in high-depth (Weirather et al., 2017). The Human cytomegalovirus (HCMV) is a ubiquitous betaherpesvirus, which can cause mononucleosis-like symptoms in adults (Cohen and Corey, 1985), and severe life-threatening infections in newborns (Wen et al., 2002). Latent HCMV infection has recently been implicated to affect cancer formation (Dziurzynski et al., 2012; Jin et al., 2014). Examining the transcriptome of the virus can go a long way in helping understand its molecular biology. Short-read RNA sequencing studies have discovered splice junctions and non-coding transcripts (Gatherer et al., 2011) and have shown that the most abundant HCMV transcripts are similarly expressed in different cell types (Cheng et al., 2017). Our long-read RNA sequencing experiments using the Pacific Biosciences (PacBio) RSII platform revealed a great number of transcript isoforms, polycistronic RNAs and transcriptional overlaps (Balázs et al., 2017a).

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September 22, 2019

Effects of antibiotic on microflora in ileum and cecum for broilers by 16S rRNA sequence analysis.

An experiment was conducted to analyze and compare the microbial composition, abundance, dynamic distribution, and functions without and with antibiotic fed to broilers. A 16S rRNA-sequencing approach was used to evaluate the bacterial composition of the gut of male broilers under different groups. A total of 240 1-day old AA male broilers were randomly assigned to two groups, with 120 broilers per group. The treatment group was administered an antibiotic with their feed, while the control group was not administered antibiotic (control group). A total of 10 replicates were assessed per treatment. The control group was fed a basal diet containing corn, soybean meal, and cottonseed meal and met the nutritional requirement. The antibiotic group was fed 100 mg/kg aureomycin (based on the basal diet). The trial lasted 42 days. Operational taxonomic unit partition and classification, alpha diversity, taxonomic composition, beta diversity, and microflora comparative analyses along with key species screening were performed for all of the treatment groups. Our data indicate that aureomycin treatment in broilers is directly correlated with variations of the gut content of specific bacterial taxa, and herein provide insights into the impact of antibiotic on microbial communities in cecum and ileum of broiler chickens.© 2018 Japanese Society of Animal Science.

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September 22, 2019

Transcriptomic study of Herpes simplex virus type-1 using full-length sequencing techniques

Herpes simplex virus type-1 (HSV-1) is a human pathogenic member of the Alphaherpesvirinae subfamily of herpesviruses. The HSV-1 genome is a large double-stranded DNA specifying about 85 protein coding genes. The latest surveys have demonstrated that the HSV-1 transcriptome is much more complex than it had been thought before. Here, we provide a long-read sequencing dataset, which was generated by using the RSII and Sequel systems from Pacific Biosciences (PacBio), as well as MinION sequencing system from Oxford Nanopore Technologies (ONT). This dataset contains 39,096 reads of inserts (ROIs) mapped to the HSV-1 genome (X14112) in RSII sequencing, while Sequel sequencing yielded 77,851 ROIs. The MinION cDNA sequencing altogether resulted in 158,653 reads, while the direct RNA-seq produced 16,516 reads. This dataset can be utilized for the identification of novel HSV RNAs and transcripts isoforms, as well as for the comparison of the quality and length of the sequencing reads derived from the currently available long- read sequencing platforms. The various library preparation approaches can also be compared with each other.

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September 22, 2019

Young genes have distinct gene structure, epigenetic profiles, and transcriptional regulation.

Species-specific, new, or “orphan” genes account for 10%-30% of eukaryotic genomes. Although initially considered to have limited function, an increasing number of orphan genes have been shown to provide important phenotypic innovation. How new genes acquire regulatory sequences for proper temporal and spatial expression is unknown. Orphan gene regulation may rely in part on origination in open chromatin adjacent to preexisting promoters, although this has not yet been assessed by genome-wide analysis of chromatin states. Here, we combine taxon-rich nematode phylogenies with Iso-Seq, RNA-seq, ChIP-seq, and ATAC-seq to identify the gene structure and epigenetic signature of orphan genes in the satellite model nematode Pristionchus pacificus Consistent with previous findings, we find young genes are shorter, contain fewer exons, and are on average less strongly expressed than older genes. However, the subset of orphan genes that are expressed exhibit distinct chromatin states from similarly expressed conserved genes. Orphan gene transcription is determined by a lack of repressive histone modifications, confirming long-held hypotheses that open chromatin is important for new gene formation. Yet orphan gene start sites more closely resemble enhancers defined by H3K4me1, H3K27ac, and ATAC-seq peaks, in contrast to conserved genes that exhibit traditional promoters defined by H3K4me3 and H3K27ac. Although the majority of orphan genes are located on chromosome arms that contain high recombination rates and repressive histone marks, strongly expressed orphan genes are more randomly distributed. Our results support a model of new gene origination by rare integration into open chromatin near enhancers.© 2018 Werner et al.; Published by Cold Spring Harbor Laboratory Press.

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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.

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September 22, 2019

Improved high-quality genome assembly and annotation of Tibetan hulless barley

Background The Tibetan hulless barley (Hordeum vulgare L. var. nudum), also called textquotedblleftQingketextquotedblright in Chinese and textquotedblleftNetextquotedblright in Tibetan, is the staple food for Tibetans and an important livestock feed in the Tibetan Plateau. The Tibetan hulless barley in China has about 3500 years of cultivation history, mainly produced in Tibet, Qinghai, Sichuan, Yunnan and other areas. In addition, Tibetan hulless barley has rich nutritional value and outstanding health effects, including the beta glucan, dietary fiber, amylopectin, the contents of trace elements, which are higher than any other cereal crops.Findings Here, we reported an improved high-quality assembly of Tibetan hulless barley genome with 4.0 Gb in size. We employed the falcon assembly package, scaffolding and error correction tools to finish improvement using PacBio long reads sequencing technology, with contig and scaffold N50 lengths of 1.563Mb and 4.006Mb, respectively, representing more continuous than the original Tibetan hulless barley genome nearly two orders of magnitude. We also re-annotated the new assembly, and reported 61,303 stringent confident putative protein-coding genes, of which 40,457 is HC genes. We have developed a new Tibetan hulless barley genome database (THBGD) to download and use friendly, as well as to better manage the information of the Tibetan hulless barley genetic resources.Conclusions The availability of new Tibetan hulless barley genome and annotations will take the genetics of Tibetan hulless barley to a new level and will greatly simplify the breeders effort. It will also enrich the granary of the Tibetan people.AbbreviationsBLASTBasic Local Alignment Search ToolBUSCOBenchmarking Universal Single-Copy OrthologsQVquality valuePacBioPacifc BiosciencesRNA-seqRNA sequencingNGSNext generation sequencingTGSThird generation sequencingTHBGDTibetan hulless barley Genome Database

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September 22, 2019

Complete genome analysis of Gluconacetobacter xylinus CGMCC 2955 for elucidating bacterial cellulose biosynthesis and metabolic regulation.

Complete genome sequence of Gluconacetobacter xylinus CGMCC 2955 for fine control of bacterial cellulose (BC) synthesis is presented here. The genome, at 3,563,314?bp, was found to contain 3,193 predicted genes without gaps. There are four BC synthase operons (bcs), among which only bcsI is structurally complete, comprising bcsA, bcsB, bcsC, and bcsD. Genes encoding key enzymes in glycolytic, pentose phosphate, and BC biosynthetic pathways and in the tricarboxylic acid cycle were identified. G. xylinus CGMCC 2955 has a complete glycolytic pathway because sequence data analysis revealed that this strain possesses a phosphofructokinase (pfk)-encoding gene, which is absent in most BC-producing strains. Furthermore, combined with our previous results, the data on metabolism of various carbon sources (monosaccharide, ethanol, and acetate) and their regulatory mechanism of action on BC production were explained. Regulation of BC synthase (Bcs) is another effective method for precise control of BC biosynthesis, and cyclic diguanylate (c-di-GMP) is the key activator of BcsA-BcsB subunit of Bcs. The quorum sensing (QS) system was found to positively regulate phosphodiesterase, which decomposed c-di-GMP. Thus, in this study, we demonstrated the presence of QS in G. xylinus CGMCC 2955 and proposed a possible regulatory mechanism of QS action on BC production.

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September 22, 2019

A single-molecule sequencing assay for the comprehensive profiling of T4 DNA ligase fidelity and bias during DNA end-joining.

DNA ligases are key enzymes in molecular and synthetic biology that catalyze the joining of breaks in duplex DNA and the end-joining of DNA fragments. Ligation fidelity (discrimination against the ligation of substrates containing mismatched base pairs) and bias (preferential ligation of particular sequences over others) have been well-studied in the context of nick ligation. However, almost no data exist for fidelity and bias in end-joining ligation contexts. In this study, we applied Pacific Biosciences Single-Molecule Real-Time sequencing technology to directly sequence the products of a highly multiplexed ligation reaction. This method has been used to profile the ligation of all three-base 5′-overhangs by T4 DNA ligase under typical ligation conditions in a single experiment. We report the relative frequency of all ligation products with or without mismatches, the position-dependent frequency of each mismatch, and the surprising observation that 5′-TNA overhangs ligate extremely inefficiently compared to all other Watson-Crick pairings. The method can easily be extended to profile other ligases, end-types (e.g. blunt ends and overhangs of different lengths), and the effect of adjacent sequence on the ligation results. Further, the method has the potential to provide new insights into the thermodynamics of annealing and the kinetics of end-joining reactions.

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September 22, 2019

Complete genome sequencing of exopolysaccharide-producing Lactobacillus plantarum K25 provides genetic evidence for the probiotic functionality and cold endurance capacity of the strain.

Lactobacillus plantarum (L. plantarum) K25 is a probiotic strain isolated from Tibetan kefir. Previous studies showed that this exopolysaccharide (EPS)-producing strain was antimicrobial active and cold tolerant. These functional traits were evidenced by complete genome sequencing of strain K25 with a circular 3,175,846-bp chromosome and six circular plasmids, encoding 3365 CDSs, 16 rRNA genes and 70 tRNA genes. Genomic analysis of L. plantarum K25 illustrates that this strain contains the previous reported mechanisms of probiotic functionality and cold tolerance, involving plantaricins, lysozyme, bile salt hydrolase, chaperone proteins, osmoprotectant, oxidoreductase, EPSs and terpenes. Interestingly, strain K25 harbors more genes that function in defense mechanisms, and lipid transport and metabolism, in comparison with other L. plantarum strains reported. The present study demonstrates the comprehensive analysis of genes related to probiotic functionalities of an EPS-producing L. plantarum strain based on whole genome sequencing.

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September 22, 2019

A rapid method for directed gene knockout for screening in G0 zebrafish.

Zebrafish is a powerful model for forward genetics. Reverse genetic approaches are limited by the time required to generate stable mutant lines. We describe a system for gene knockout that consistently produces null phenotypes in G0 zebrafish. Yolk injection of sets of four CRISPR/Cas9 ribonucleoprotein complexes redundantly targeting a single gene recapitulated germline-transmitted knockout phenotypes in >90% of G0 embryos for each of 8 test genes. Early embryonic (6 hpf) and stable adult phenotypes were produced. Simultaneous multi-gene knockout was feasible but associated with toxicity in some cases. To facilitate use, we generated a lookup table of four-guide sets for 21,386 zebrafish genes and validated several. Using this resource, we targeted 50 cardiomyocyte transcriptional regulators and uncovered a role of zbtb16a in cardiac development. This system provides a platform for rapid screening of genes of interest in development, physiology, and disease models in zebrafish. Copyright © 2018 Elsevier Inc. All rights reserved.

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September 22, 2019

Genomics of Corynebacterium striatum, an emerging multidrug-resistant pathogen of immunocompromised patients.

Corynebacterium striatum is an emerging multidrug-resistant (MDR) pathogen of immunocompromised and chronically ill patients. The objective of these studies was to provide a detailed genomic analysis of disease-causing C. striatum and determine the genomic drivers of resistance and resistance-gene transmission.A multi-institutional and prospective pathogen genomics programme flagged seven MDR C. striatum infections occurring close in time, and specifically in immunocompromised patients with underlying respiratory diseases. Whole genome sequencing was used to identify clonal relationships among strains, genetic causes of antimicrobial resistance, and their mobilization capacity. Matrix-assisted linear desorption/ionization-time-of-flight analyses of sequenced isolates provided curated content to improve rapid clinical identification in subsequent cases.Epidemiological and genomic analyses identified a related cluster of three out of seven C. striatum among lung transplant patients who had common procedures and exposures at an outlying institution. Genomic analyses further elucidated drivers of the MDR phenotypes, including resistance genes mobilized by IS3504 and ISCg9a-like insertion sequences. Seven mobilizable resistance genes were localized to a common chromosomal region bounded by unpaired insertion sequences, suggesting that a single recombination event could spread resistance to aminoglycosides, macrolides, lincosamides and tetracyclines to naive strains.In-depth genomic studies of MDR C. striatum reveal its capacity for clonal spread within and across healthcare institutions and identify novel vectors that can mobilize multiple forms of drug resistance, further complicating efforts to treat infections in immunocompromised populations. Copyright © 2018 European Society of Clinical Microbiology and Infectious Diseases. All rights reserved.

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