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September 22, 2019  |  

Comparison of the mitochondrial genomes and steady state transcriptomes of two strains of the trypanosomatid parasite, Leishmania tarentolae.

U-insertion/deletion RNA editing is a post-transcriptional mitochondrial RNA modification phenomenon required for viability of trypanosomatid parasites. Small guide RNAs encoded mainly by the thousands of catenated minicircles contain the information for this editing. We analyzed by NGS technology the mitochondrial genomes and transcriptomes of two strains, the old lab UC strain and the recently isolated LEM125 strain. PacBio sequencing provided complete minicircle sequences which avoided the assembly problem of short reads caused by the conserved regions. Minicircles were identified by a characteristic size, the presence of three short conserved sequences, a region of inherently bent DNA and the presence of single gRNA genes at a fairly defined location. The LEM125 strain contained over 114 minicircles encoding different gRNAs and the UC strain only ~24 minicircles. Some LEM125 minicircles contained no identifiable gRNAs. Approximate copy numbers of the different minicircle classes in the network were determined by the number of PacBio CCS reads that assembled to each class. Mitochondrial RNA libraries from both strains were mapped against the minicircle and maxicircle sequences. Small RNA reads mapped to the putative gRNA genes but also to multiple regions outside the genes on both strands and large RNA reads mapped in many cases over almost the entire minicircle on both strands. These data suggest that minicircle transcription is complete and bidirectional, with 3′ processing yielding the mature gRNAs. Steady state RNAs in varying abundances are derived from all maxicircle genes, including portions of the repetitive divergent region. The relative extents of editing in both strains correlated with the presence of a cascade of cognate gRNAs. These data should provide the foundation for a deeper understanding of this dynamic genetic system as well as the evolutionary variation of editing in different strains.

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September 22, 2019  |  

Genome-wide transcriptome profiling of the medicinal plant Zanthoxylum planispinum using a single-molecule direct RNA sequencing approach.

High-throughput RNA sequencing has revolutionized transcriptome-based studies of candidate genes, key pathways and gene regulation in non-model organisms. We analyzed full-length cDNA sequences in Zanthoxylum planispinum (Z. planispinum), a medicinal herb in major parts of East Asia. The full-length mRNA derived from tissues of leaf, early fruit and maturing fruit stage were sequenced using PacBio RSII platform to identify isoform transcriptome. We obtained 51,402 unigenes, with average 1781?bp per gene in 82.473?Mb gene lengths. Among 51,402, 3963 unigenes showed variety of isoform. By selection of one representative gene among each of the various isoforms, we finalized 46,306 unique gene set for this herb. We identified 76 cytochrome P450 (CYP450) and related isoforms that are of the wide diversity in the molecular function and biological process. These transcriptome data of Z. planispinum will provide a good resource to study metabolic engineering for the production of valuable medicinal drugs and phytochemicals. Copyright © 2018. Published by Elsevier Inc.

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September 22, 2019  |  

PhyloPythiaS+: a self-training method for the rapid reconstruction of low-ranking taxonomic bins from metagenomes.

Background. Metagenomics is an approach for characterizing environmental microbial communities in situ, it allows their functional and taxonomic characterization and to recover sequences from uncultured taxa. This is often achieved by a combination of sequence assembly and binning, where sequences are grouped into ‘bins’ representing taxa of the underlying microbial community. Assignment to low-ranking taxonomic bins is an important challenge for binning methods as is scalability to Gb-sized datasets generated with deep sequencing techniques. One of the best available methods for species bins recovery from deep-branching phyla is the expert-trained PhyloPythiaS package, where a human expert decides on the taxa to incorporate in the model and identifies ‘training’ sequences based on marker genes directly from the sample. Due to the manual effort involved, this approach does not scale to multiple metagenome samples and requires substantial expertise, which researchers who are new to the area do not have. Results. We have developed PhyloPythiaS+, a successor to our PhyloPythia(S) software. The new (+) component performs the work previously done by the human expert. PhyloPythiaS+ also includes a new k-mer counting algorithm, which accelerated the simultaneous counting of 4-6-mers used for taxonomic binning 100-fold and reduced the overall execution time of the software by a factor of three. Our software allows to analyze Gb-sized metagenomes with inexpensive hardware, and to recover species or genera-level bins with low error rates in a fully automated fashion. PhyloPythiaS+ was compared to MEGAN, taxator-tk, Kraken and the generic PhyloPythiaS model. The results showed that PhyloPythiaS+ performs especially well for samples originating from novel environments in comparison to the other methods. Availability. PhyloPythiaS+ in a virtual machine is available for installation under Windows, Unix systems or OS X on: https://github.com/algbioi/ppsp/wiki.

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September 22, 2019  |  

Koumiss consumption alleviates symptoms of patients with chronic atrophic gastritis: A possible link To modulation of gut microbiota

Intestinal dysbiosisis closely related to a variety of medical conditions, especially gastrointestinal diseases. The present study aimed to investigate the effects of koumiss on chronic atrophic gastritis (CAG) in an out-patient clinical trial (n = 10; all female subjects aged 41-55; body mass index ranging from 19.5 to 25.8). Each patient consumed three servings of koumiss per day (i.e. 250 ml daily before each of 3 meals) for a 60-day period. The improvement of patients’ symptoms was monitored by comparing the total scores of symptoms before and after the treatment. Meanwhile, the changes in the patients’ fecal microbiota composition and specific blood parameters were determined. After the 60-day koumiss administration, significant symptom improvements were observed, as evidenced by the reduction of the total symptoms score, and changes in blood platelet and cholesterol levels. The changes in patients’ fecal microbiota composition were found. The patients’ fecal microbiota fell into two distinct enterotypes, Bacteroides dorei/ Bacteroides uniformis (BB-enterotype) and Prevotella copri (P-enterotype). Significant less Bacteroides uniformis was found in the BB-enterotype patient group, while significant more butyrate-producing bacteria (e.g. Eubacterium rectale and Faecalibacterium prausnitzii) were found in the P-enterotype patient group, following koumiss administration. After stopping koumiss consumption, the relative abundance of some biomarker taxa returned to the original level, suggesting that the gut microbiota modulatory effect was not permanent and that continuous koumiss administration was required to maintain the therapeutic effect. In conclusion, koumiss consumption could alleviate the symptoms of CAG patients. Our results may help understand the mechanism of koumiss in alleviating CAG disease symptoms, facilitating the development of such products with desired therapeutic functions.

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September 22, 2019  |  

Elevated expression of a minor isoform of ANK3 is a risk factor for bipolar disorder.

Ankyrin-3 (ANK3) is one of the few genes that have been consistently identified as associated with bipolar disorder by multiple genome-wide association studies. However, the exact molecular basis of the association remains unknown. A rare loss-of-function splice-site SNP (rs41283526*G) in a minor isoform of ANK3 (incorporating exon ENSE00001786716) was recently identified as protective of bipolar disorder and schizophrenia. This suggests that an elevated expression of this isoform may be involved in the etiology of the disorders. In this study, we used novel approaches and data sets to test this hypothesis. First, we strengthen the statistical evidence supporting the allelic association by replicating the protective effect of the minor allele of rs41283526 in three additional large independent samples (meta-analysis p-values: 6.8E-05 for bipolar disorder and 8.2E-04 for schizophrenia). Second, we confirm the hypothesis that both bipolar and schizophrenia patients have a significantly higher expression of this isoform than controls (p-values: 3.3E-05 for schizophrenia and 9.8E-04 for bipolar type I). Third, we determine the transcription start site for this minor isoform by Pacific Biosciences sequencing of full-length cDNA and show that it is primarily expressed in the corpus callosum. Finally, we combine genotype and expression data from a large Norwegian sample of psychiatric patients and controls, and show that the risk alleles in ANK3 identified by bipolar disorder GWAS are located near the transcription start site of this isoform and are significantly associated with its elevated expression. Together, these results point to the likely molecular mechanism underlying ANK3´s association with bipolar disorder.

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September 22, 2019  |  

A comprehensive benchmarking study of protocols and sequencing platforms for 16S rRNA community profiling.

In the last 5 years, the rapid pace of innovations and improvements in sequencing technologies has completely changed the landscape of metagenomic and metagenetic experiments. Therefore, it is critical to benchmark the various methodologies for interrogating the composition of microbial communities, so that we can assess their strengths and limitations. The most common phylogenetic marker for microbial community diversity studies is the 16S ribosomal RNA gene and in the last 10 years the field has moved from sequencing a small number of amplicons and samples to more complex studies where thousands of samples and multiple different gene regions are interrogated.We assembled 2 synthetic communities with an even (EM) and uneven (UM) distribution of archaeal and bacterial strains and species, as metagenomic control material, to assess performance of different experimental strategies. The 2 synthetic communities were used in this study, to highlight the limitations and the advantages of the leading sequencing platforms: MiSeq (Illumina), The Pacific Biosciences RSII, 454 GS-FLX/+ (Roche), and IonTorrent (Life Technologies). We describe an extensive survey based on synthetic communities using 3 experimental designs (fusion primers, universal tailed tag, ligated adaptors) across the 9 hypervariable 16S rDNA regions. We demonstrate that library preparation methodology can affect data interpretation due to different error and chimera rates generated during the procedure. The observed community composition was always biased, to a degree that depended on the platform, sequenced region and primer choice. However, crucially, our analysis suggests that 16S rRNA sequencing is still quantitative, in that relative changes in abundance of taxa between samples can be recovered, despite these biases.We have assessed a range of experimental conditions across several next generation sequencing platforms using the most up-to-date configurations. We propose that the choice of sequencing platform and experimental design needs to be taken into consideration in the early stage of a project by running a small trial consisting of several hypervariable regions to quantify the discriminatory power of each region. We also suggest that the use of a synthetic community as a positive control would be beneficial to identify the potential biases and procedural drawbacks that may lead to data misinterpretation. The results of this study will serve as a guideline for making decisions on which experimental condition and sequencing platform to consider to achieve the best microbial profiling.

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September 22, 2019  |  

Indoleacrylic acid produced by commensal Peptostreptococcus species suppresses inflammation.

Host factors in the intestine help select for bacteria that promote health. Certain commensals can utilize mucins as an energy source, thus promoting their colonization. However, health conditions such as inflammatory bowel disease (IBD) are associated with a reduced mucus layer, potentially leading to dysbiosis associated with this disease. We characterize the capability of commensal species to cleave and transport mucin-associated monosaccharides and identify several Clostridiales members that utilize intestinal mucins. One such mucin utilizer, Peptostreptococcus russellii, reduces susceptibility to epithelial injury in mice. Several Peptostreptococcus species contain a gene cluster enabling production of the tryptophan metabolite indoleacrylic acid (IA), which promotes intestinal epithelial barrier function and mitigates inflammatory responses. Furthermore, metagenomic analysis of human stool samples reveals that the genetic capability of microbes to utilize mucins and metabolize tryptophan is diminished in IBD patients. Our data suggest that stimulating IA production could promote anti-inflammatory responses and have therapeutic benefits. Copyright © 2017 Elsevier Inc. All rights reserved.

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September 22, 2019  |  

Interaction between the microbiome and TP53 in human lung cancer.

Lung cancer is the leading cancer diagnosis worldwide and the number one cause of cancer deaths. Exposure to cigarette smoke, the primary risk factor in lung cancer, reduces epithelial barrier integrity and increases susceptibility to infections. Herein, we hypothesize that somatic mutations together with cigarette smoke generate a dysbiotic microbiota that is associated with lung carcinogenesis. Using lung tissue from 33 controls and 143 cancer cases, we conduct 16S ribosomal RNA (rRNA) bacterial gene sequencing, with RNA-sequencing data from lung cancer cases in The Cancer Genome Atlas serving as the validation cohort.Overall, we demonstrate a lower alpha diversity in normal lung as compared to non-tumor adjacent or tumor tissue. In squamous cell carcinoma specifically, a separate group of taxa are identified, in which Acidovorax is enriched in smokers. Acidovorax temporans is identified within tumor sections by fluorescent in situ hybridization and confirmed by two separate 16S rRNA strategies. Further, these taxa, including Acidovorax, exhibit higher abundance among the subset of squamous cell carcinoma cases with TP53 mutations, an association not seen in adenocarcinomas.The results of this comprehensive study show both microbiome-gene and microbiome-exposure interactions in squamous cell carcinoma lung cancer tissue. Specifically, tumors harboring TP53 mutations, which can impair epithelial function, have a unique bacterial consortium that is higher in relative abundance in smoking-associated tumors of this type. Given the significant need for clinical diagnostic tools in lung cancer, this study may provide novel biomarkers for early detection.

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September 22, 2019  |  

A single-cell genome for Thiovulum sp.

We determined a significant fraction of the genome sequence of a representative of Thiovulum, the uncultivated genus of colorless sulfur Epsilonproteobacteria, by analyzing the genome sequences of four individual cells collected from phototrophic mats from Elkhorn Slough, California. These cells were isolated utilizing a microfluidic laser-tweezing system, and their genomes were amplified by multiple-displacement amplification prior to sequencing. Thiovulum is a gradient bacterium found at oxic-anoxic marine interfaces and noted for its distinctive morphology and rapid swimming motility. The genomic sequences of the four individual cells were assembled into a composite genome consisting of 221 contigs covering 2.083 Mb including 2,162 genes. This single-cell genome represents a genomic view of the physiological capabilities of isolated Thiovulum cells. Thiovulum is the second-fastest bacterium ever observed, swimming at 615 µm/s, and this genome shows that this rapid swimming motility is a result of a standard flagellar machinery that has been extensively characterized in other bacteria. This suggests that standard flagella are capable of propelling bacterial cells at speeds much faster than typically thought. Analysis of the genome suggests that naturally occurring Thiovulum populations are more diverse than previously recognized and that studies performed in the past probably address a wide range of unrecognized genotypic and phenotypic diversities of Thiovulum. The genome presented in this article provides a basis for future isolation-independent studies of Thiovulum, where single-cell and metagenomic tools can be used to differentiate between different Thiovulum genotypes.

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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.

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September 22, 2019  |  

Minimap2: pairwise alignment for nucleotide sequences.

Recent advances in sequencing technologies promise ultra-long reads of ~100 kb in average, full-length mRNA or cDNA reads in high throughput and genomic contigs over 100 Mb in length. Existing alignment programs are unable or inefficient to process such data at scale, which presses for the development of new alignment algorithms.Minimap2 is a general-purpose alignment program to map DNA or long mRNA sequences against a large reference database. It works with accurate short reads of =100?bp in length, =1?kb genomic reads at error rate ~15%, full-length noisy Direct RNA or cDNA reads and assembly contigs or closely related full chromosomes of hundreds of megabases in length. Minimap2 does split-read alignment, employs concave gap cost for long insertions and deletions and introduces new heuristics to reduce spurious alignments. It is 3-4 times as fast as mainstream short-read mappers at comparable accuracy, and is =30 times faster than long-read genomic or cDNA mappers at higher accuracy, surpassing most aligners specialized in one type of alignment.https://github.com/lh3/minimap2.Supplementary data are available at Bioinformatics online.

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September 22, 2019  |  

Transcriptome sequencing reveals thousands of novel long non-coding RNAs in B cell lymphoma.

Gene profiling of diffuse large B cell lymphoma (DLBCL) has revealed broad gene expression deregulation compared to normal B cells. While many studies have interrogated well known and annotated genes in DLBCL, none have yet performed a systematic analysis to uncover novel unannotated long non-coding RNAs (lncRNA) in DLBCL. In this study we sought to uncover these lncRNAs by examining RNA-seq data from primary DLBCL tumors and performed supporting analysis to identify potential role of these lncRNAs in DLBCL.We performed a systematic analysis of novel lncRNAs from the poly-adenylated transcriptome of 116 primary DLBCL samples. RNA-seq data were processed using de novo transcript assembly pipeline to discover novel lncRNAs in DLBCL. Systematic functional, mutational, cross-species, and co-expression analyses using numerous bioinformatics tools and statistical analysis were performed to characterize these novel lncRNAs.We identified 2,632 novel, multi-exonic lncRNAs expressed in more than one tumor, two-thirds of which are not expressed in normal B cells. Long read single molecule sequencing supports the splicing structure of many of these lncRNAs. More than one-third of novel lncRNAs are differentially expressed between the two major DLBCL subtypes, ABC and GCB. Novel lncRNAs are enriched at DLBCL super-enhancers, with a fraction of them conserved between human and dog lymphomas. We see transposable elements (TE) overlap in the exonic regions; particularly significant in the last exon of the novel lncRNAs suggest potential usage of cryptic TE polyadenylation signals. We identified highly co-expressed protein coding genes for at least 88 % of the novel lncRNAs. Functional enrichment analysis of co-expressed genes predicts a potential function for about half of novel lncRNAs. Finally, systematic structural analysis of candidate point mutations (SNVs) suggests that such mutations frequently stabilize lncRNA structures instead of destabilizing them.Discovery of these 2,632 novel lncRNAs in DLBCL significantly expands the lymphoma transcriptome and our analysis identifies potential roles of these lncRNAs in lymphomagenesis and/or tumor maintenance. For further studies, these novel lncRNAs also provide an abundant source of new targets for antisense oligonucleotide pharmacology, including shared targets between human and dog lymphomas.

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September 22, 2019  |  

Comparative genomics reveals cotton-specific virulence factors in flexible genomic regions in Verticillium dahliae and evidence of horizontal gene transfer from Fusarium.

Verticillium dahliae isolates are most virulent on the host from which they were originally isolated. Mechanisms underlying these dominant host adaptations are currently unknown. We sequenced the genome of V. dahliae Vd991, which is highly virulent on its original host, cotton, and performed comparisons with the reference genomes of JR2 (from tomato) and VdLs.17 (from lettuce). Pathogenicity-related factor prediction, orthology and multigene family classification, transcriptome analyses, phylogenetic analyses, and pathogenicity experiments were performed. The Vd991 genome harbored several exclusive, lineage-specific (LS) genes within LS regions (LSRs). Deletion mutants of the seven genes within one LSR (G-LSR2) in Vd991 were less virulent only on cotton. Integration of G-LSR2 genes individually into JR2 and VdLs.17 resulted in significantly enhanced virulence on cotton but did not affect virulence on tomato or lettuce. Transcription levels of the seven LS genes in Vd991 were higher during the early stages of cotton infection, as compared with other hosts. Phylogenetic analyses suggested that G-LSR2 was acquired from Fusarium oxysporum f. sp. vasinfectum through horizontal gene transfer. Our results provide evidence that horizontal gene transfer from Fusarium to Vd991 contributed significantly to its adaptation to cotton and may represent a significant mechanism in the evolution of an asexual plant pathogen.© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

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September 22, 2019  |  

Sooty mangabey genome sequence provides insight into AIDS resistance in a natural SIV host.

In contrast to infections with human immunodeficiency virus (HIV) in humans and simian immunodeficiency virus (SIV) in macaques, SIV infection of a natural host, sooty mangabeys (Cercocebus atys), is non-pathogenic despite high viraemia. Here we sequenced and assembled the genome of a captive sooty mangabey. We conducted genome-wide comparative analyses of transcript assemblies from C. atys and AIDS-susceptible species, such as humans and macaques, to identify candidates for host genetic factors that influence susceptibility. We identified several immune-related genes in the genome of C. atys that show substantial sequence divergence from macaques or humans. One of these sequence divergences, a C-terminal frameshift in the toll-like receptor-4 (TLR4) gene of C. atys, is associated with a blunted in vitro response to TLR-4 ligands. In addition, we found a major structural change in exons 3-4 of the immune-regulatory protein intercellular adhesion molecule 2 (ICAM-2); expression of this variant leads to reduced cell surface expression of ICAM-2. These data provide a resource for comparative genomic studies of HIV and/or SIV pathogenesis and may help to elucidate the mechanisms by which SIV-infected sooty mangabeys avoid AIDS.

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