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Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2018

Comprehensive analysis of small RNA profile by massive parallel sequencing in HTLV-1 asymptomatic subjects with monoclonal and polyclonal rearrangement of the T-cell antigen receptor γ-chain

IntroductionIn this study, we used a massive parallel sequencing technology to investigate the cellular small RNA (sRNA) operating in peripheral blood mononuclear cells (PBMCs) of the Human T-lymphotropic virus type I (HTLV-I) infected asymptomatic subjects with a monoclonal and polyclonal rearrangement of the T-cell antigen receptor {gamma}-chain.\n\nMaterials and MethodsBlood samples from 15 HTLV-1 asymptomatic carriers who were tested for clonal TCR-{gamma} gene (seven and eight subjects presented monoclonal and polyclonal expansion of HTLV-1 infected cells, respectively), and were submitted to Illumina for small RNA library construction. sRNA libraries were prepared from cryoperserved PBMCs using TrueSeq Small RNA Library Preparation Kit (Illumina). The sRNA-Seq reads were aligned, annotated, and profiled by different bioinformatics tools.\n\nResultsThrough bioinformatics analysis, we identified a total of 494 known sRNAs and 120 putative novel sRNAs. Twenty-two known and 15 novel sRNA showed a different expression (>2-fold) between the asymptomatic monoclonal (ASM) and asymptomatic polyclonal carriers (ASP). The hsa-mir-196a-5p was the most abundantly upregulated micro RNA (miRNA) and the hsa-mir-133a followed by hsa-mir-509-3p were significantly downregulated miRNAs with more than a three-fold difference in the ASM than ASP group. The target genes predicted to be regulated by the differentially expressed miRNAs play essential roles in diverse biological processes including cell proliferation, differentiation, and/or apoptosis.\n\nDiscussionOur results provide an opportunity for a further understanding of sRNA regulation and function in HTLV-1 infected subjects with monoclonality evidence.

microbiology↗

bioRxiv2018

Nach is a novel ancestral subfamily of the CNC-bZIP transcription factors selected during evolution from the marine bacteria to human

All living organisms have undergone the evolutionary selection under the changing natural environments to survive as diverse life forms. All life processes including normal homeostatic development and growth into organismic bodies with distinct cellular identifications, as well as their adaptive responses to various intracellular and environmental stresses, are tightly controlled by signaling of transcriptional networks towards regulation of cognate genes by many different transcription factors. Amongst them, one of the most conserved is the basic-region leucine zipper (bZIP) family. They play vital roles essential for cell proliferation, differentiation and maintenance in complex multicellular organisms. Notably, an unresolved divergence on the evolution of bZIP proteins is addressed here. By a combination of bioinformatics with genomics and molecular biology, we have demonstrated that two of the most ancestral family members classified into BATF and Jun subgroups are originated from viruses, albeit expansion and diversification of the bZIP superfamily occur in different vertebrates. Interestingly, a specific ancestral subfamily of bZIP proteins is identified and also designated Nach (Nrf and CNC homology) on account of their highly conservativity with NF-E2 p45 subunit-related factors Nrf1/2. Further experimental evidence reveals that Nach1/2 from the marine bacteria exerts distinctive functions from Nrf1/2 in the transcriptional ability to regulate antioxidant response element (ARE)-driven cytoprotective genes. Collectively, an insight into Nach/CNC-bZIP proteins provides a better understanding of distinct biological functions between these factors selected during evolution from the marine bacteria to human.\n\nSignificanceWe identified the novel ancestral subfamily (i.e. Nach) of CNC-bZIP transcription factors with highly conservativity from marine bacteria to human. Combination of bioinformatics with genomics and molecular biology demonstrated that two of the most ancestral family members classified into BATF and Jun subgroups are originated from viruses. The Jun and CNC subfamilies also share a common origin of these bZIP proteins. Further experimental evidence reveals that Nach1/2 from the marine bacteria exerts nuance functions from human Nrf1/2 in the transcriptional ability to regulate antioxidant response element (ARE)-driven genes, responsible for the host cytoprotection against inflammation and cancer. Overall, this study is of multidisciplinary interests to provide a better understanding of distinct biological functions between Nach/CNC-bZIPs selected during evolution.

ecology↗

bioRxiv2018

Mono-homologous linear DNA recombination by the non-homologous end-joining pathway as a novel and simple gene inactivation method: a proof of concept study in Dietzia sp. DQ12-45-1b

Non-homologous end-joining (NHEJ) is critical for genome stability because of its roles in double-strand break repair. Ku and ligase D (LigD) are the crucial proteins in this process, and strains expressing Ku and LigD can cyclize linear DNA in vivo. Herein, we established a proof-of-concept mono-homologous linear DNA recombination for gene inactivation or genome editing by which cyclization of linear DNA in vivo by NHEJ could be used to generate non-replicable circular DNA and could allow allelic exchanges between the circular DNA and the chromosome. We achieved this approach in Dietzia sp. DQ12-45-1b, which expresses Ku and LigD homologs and presents NHEJ activity. By transforming the strain with a linear DNA mono homolog to the sequence in chromosome, we mutated the genome. This method did not require the screening of suitable plasmids and was easy and time-effective. Bioinformatic analysis showed that more than 20% prokaryotic organisms contain Ku and LigD, suggesting the wide distribution of NHEJ activities. Moreover, the Escherichia coli strain also showed NHEJ activity when the Ku and LigD of Dietzia sp. DQ12-45-1b were introduced and expressed in it. Therefore, this method may be a widely applicable genome editing tool for diverse prokaryotic organisms, especially for non-model microorganisms.\n\nIMPORTANCEThe non-model gram-positive bacteria lack efficient genetic manipulation systems, but they express genes encoding Ku and LigD. The NHEJ pathway in Dietzia sp. DQ12-45-1b was evaluated and was used to successfully knockout eleven genes in the genome. Since bioinformatic studies revealed that the putative genes encoding Ku and LigD ubiquitously exist in phylogenetically diverse bacteria and archaea, the mono-homologous linear DNA recombination by the NHEJ pathway could be a potentially applicable genetic manipulation method for diverse non-model prokaryotic organisms.

microbiology↗

bioRxiv2018

FusoPortal: An interactive repository of hybrid MinION sequenced Fusobacterium genomes improves gene identification and characterization

Here we present FusoPortal, an interactive repository of Fusobacterium genomes that were sequenced using a hybrid MinION long-read sequencing pipeline, followed by assembly and annotation using a diverse portfolio of predominantly open-source software. Significant efforts were made to provide genomic and bioinformatic data as downloadable files, including raw sequencing reads, genome maps, gene annotations, protein functional analysis and classifications, and a custom BLAST server for FusoPortal genomes. FusoPortal has been initiated with eight complete genomes, of which seven were previously only drafts that varied from 24-67 contigs. We showcase that genomes in FusoPortal provide accurate open reading frame annotations, and have corrected a number of large genes (>3 kb) that were previously misannotated due to contig boundaries. In summary, FusoPortal (http://fusoportal.org) is the first database of MinION sequenced and completely assembled Fusobacterium genomes, and this central Fusobacterium genomic and bioinformatic resource will aid the scientific community in developing a deeper understanding of how this human pathogen contributes to an array of diseases including periodontitis and colorectal cancer.\n\nImportanceIn this study, we report a hybrid MinION whole genome sequencing pipeline, and describe the genomic characteristics of the first eight strains deposited in the FusoPortal database. This collection of highly accurate and complete genomes drastically improves upon previous multi-contig assemblies by correcting and newly identifying a significant number of open reading frames. We believe this resource will result in the discovery of proteins and molecular mechanisms used by an oral pathogen, with the potential to further our understanding of how F. nucleatum contributes to a repertoire of diseases including periodontitis, pre-term birth, and colorectal cancer

microbiology↗

bioRxiv2018

In Silico Analysis Reveals a Shared Immune Signature in CASP8-Mutated Carcinomas with Varying Correlations to Prognosis.

BackgroundSequencing studies across multiple cancers continue to reveal the spectrum of mutations and genes involved in the pathobiology of these cancers. Exome sequencing of oral cancers, a subset of Head and Neck Squamous cell Carcinomas (HNSCs) common among tobacco-chewing populations, revealed that ~34% of the affected patients harbor mutations in the CASP8 gene. Uterine Corpus Endometrial Carcinoma (UCEC) is another cancer type where about 10% cases harbor CASP8 mutations. Caspase-8, the protease encoded by CASP8 gene, plays a dual role in programmed cell death, which in turn has an important role in tumor cell death and drug resistance. CASP8 is a protease required for the extrinsic pathway of apoptosis and is also a negative regulator of necroptosis. Using bioinformatics approaches to mine data in The Cancer Genome Atlas, we compared the molecular features and survival of these carcinomas with and without CASP8 mutations.\n\nResultsOur in silico analyses showed that HNSCs with CASP8 mutations displayed a prominent signature of genes involved in immune response and inflammation, and were rich in immune cell infiltrates. However, in contrast to Human Papilloma Virus-positive HNSCs, a subtype that exhibits high immune cell infiltration and better overall survival, HNSC patients with mutant-CASP8 tumors did not display any survival advantage. A similar bioinformatic analyses in UCECs revealed that while UCECs with CASP8 mutations also displayed an immune signature, they had better overall survival, in contrast to the HNSC scenario. On further examination, we found that there was significant up-regulation of neutrophils as well as the cytokine, IL33 in mutant-CASP8 HNSCs, both of which were not observed in mutant-CASP8 UCECs.\n\nConclusionsThese results suggested that carcinomas with mutant CASP8 have broadly similar immune signatures albeit with different effects on survival. We hypothesize that subtle tissue-dependent differences could influence survival by modifying the micro-environment of mutant-CASP8 carcinomas. High neutrophil numbers, which is a well-known negative prognosticator in HNSCs, and/or high IL33 levels may be some of the factors affecting survival of mutant-CASP8 cases.

cancer biology↗

bioRxiv2018

Stabilized Independent Component Analysis outperforms other methods in finding reproducible signals in tumoral transcriptomes

MotivationMatrix factorization methods are widely exploited in order to reduce dimensionality of transcriptomic datasets to the action of few hidden factors (metagenes). Applying such methods to similar independent datasets should yield reproducible inter-series outputs, though it was never demonstrated yet.\n\nResultsWe systematically test state-of-art methods of matrix factorization on several transcriptomic datasets of the same cancer type. Inspired by concepts of evolutionary bioinformatics, we design a new framework based on Reciprocally Best Hit (RBH) graphs in order to benchmark the methods reproducibility. We show that a particular protocol of application of Independent Component Analysis (ICA), accompanied by a stabilisation procedure, leads to a significant increase in the inter-series output reproducibility. Moreover, we show that the signals detected through this method are systematically more interpretable than those of other state-of-art methods. We developed a user-friendly tool BIODICA for performing the Stabilized ICA-based RBH meta-analysis. We apply this methodology to the study of colorectal cancer (CRC) for which 14 independent publicly available transcriptomic datasets can be collected. The resulting RBH graph maps the landscape of interconnected factors that can be associated to biological processes or to technological artefacts. These factors can be used as clinical biomarkers or robust and tumor-type specific transcriptomic signatures of tumoral cells or tumoral microenvironment. Their intensities in different samples shed light on the mechanistic basis of CRC molecular subtyping.\n\nAvailabilityThe BIODICA tool is available from https://github.com/LabBandSB/BIODICA.\n\nContactlaura.cantini@curie.fr and andrei.zinovyev@curie.fr\n\nSupplementary informationSupplementary data are available at Bioinformatics online.

systems biology↗

bioRxiv2018

High-throughput identification and marker development of perfect SSR for cultivated genus of passion fruit (Passiflora edulis)

Simple sequence repeat (SSR) markers are characterized by high polymorphism, good reproducibility and co-dominance etc. They can be easily applied to develop efficient, simple and practical molecular markers. In the present study, bioinformatics methods were applied to identify high-throughput perfect SSRs of cultivar Passiflora genome. A total of 13104 perfect SSRs were obtained. SSR core sequence structure is mainly 2-4 bases, the maximum numbers are TA, AT, TC and AG. The maximum numbers of repetitions were up to 20 times. A total of 12934 pairs of SSR markers were developed by using bioinformatics software, and 20 pairs of markers were selected for amplification specificity assessment of MTX and WJ10, and the polymorphism rate was as high as 60%. The large-scale development of the SSR markers of Passiflora cultivar has paved a foundation for the efficient utilization of the germplasm resources of passion fruit, genetic improvement of the varieties and molecular breeding.

genomics↗

bioRxiv2018

Molecular analysis of long non-coding RNA GAS5 and microRNA-34a expression signature in common solid tumors: A pilot study

Accumulating evidence indicates that non-coding RNAs including microRNAs (miRs) and long non-coding RNAs (lncRNAs) are aberrantly expressed in cancer, providing promising biomarkers for diagnosis, prognosis and/or therapeutic targets. We aimed in the current work to quantify the expression profile of miR-34a and one of its bioinformatically selected partner lncRNA growth arrest-specific 5 (GAS5) in a sample of Egyptian cancer patients, including three prevalent types of cancer in our region; renal cell carcinoma (RCC), hepatocellular carcinoma (HCC) and glioblastoma (GB) as well as to correlate these expression profiles with the available clinicopathological data in an attempt to clarify their roles in cancer. Quantitative real-time polymerase chain reaction analysis was applied. Different bioinformatics databases were searched to confirm the potential miRNAs-lncRNA interactions of the selected ncRNAs in cancer pathogenesis. GAS5 was significantly under-expressed in the three types of cancer. However, levels of miR-34a greatly varied according to the tumor type; it displayed an increased expression in RCC [4.05 (1.003-22.69), p <0.001] and a decreased expression in GB [0.35 (0.04-0.95), p <0.001]. A weak negative correlation was observed between levels of GAS5 and miR-34a in GB [r = -0.39, p =0.006]. Univariate analyses revealed a correlation of GAS5 downregulation with poor disease-free survival (r = 0.31, p =0.018) and overall survival (r = 0.28, p =0.029) in RCC but not in GB, and a marginal significance correlation with a higher number of lesions in HCC. Hierarchical clustering analysis showed RCC patients among others, could be clustered by GAS5 and miR-34a co-expression profile. Our results confirm the tumor suppressor role of GAS5 in cancer and suggest its potential applicability to be a predictor of bad outcomes with other conventional markers for various types of cancer. Further functional validation studies are warranted to confirm miR-34a/GAS5 interplay in cancer.

molecular biology↗

bioRxiv2018

Combining mathematical and statistical modeling to simulate time course bulk and single cell gene expression data in cancer with CancerInSilico

Bioinformatics techniques to analyze time course bulk and single cell omics data are advancing. The absence of a known ground truth of the dynamics of molecular changes challenges benchmarking their performance on real data. Realistic simulated time-course datasets are essential to assess the performance of time course bioinformatics algorithms. We develop an R/Bioconductor package, CancerInSilico, to simulate bulk and single cell transcriptional data from a known ground truth obtained from mathematical models of cellular systems. This package contains a general R infrastructure for running cell-based models and simulating gene expression data based on the model states. We show how to use this package to simulate a gene expression data set and consequently benchmark analysis methods on this data set with a known ground truth. The package is freely available via Bioconductor: http://bioconductor.org/packages/CancerInSilico/

systems biology↗

bioRxiv2018

Identification of a Nocardia seriolae secreted protein targeting host cell mitochondria and inducing apoptosis in fathead minnow (FHM) cells

Nocardia seriolae, is a Gram-positive, partially acid-fast, aerobic, and filamentous bacterium. This bacterium is the main pathogen of fish nocardiosis. A bioinformatic analysis based on the genomic sequence of the N. seriolae strain ZJ0503 showed that ORF3141 encoded a secreted protein with a signal peptide at the N-terminate which may target the mitochondria in the host cell. However, the functions of this protein and its homologs remain unknown. In this study, we experimentally tested the bioinformatic prediction on this protein. Mass spectrometry analysis of the extracellular products from N. seriolae showed that ORF3141 was a secreted protein. Subcellular localization of the ORF3141-GFP fusion protein revealed that the green fluorescence protein co-localized with the mitochondria, while ORF3141{Delta}sig-GFP (with the signal peptide deleted) fusion protein was evenly distributed in the whole cell of fathead minnow (FHM) cells. Thus, the N-terminate signal peptide had a significant impact on mitochondrial targeting. Notably, the expression of ORF3141 protein changed the distribution of mitochondria from perinuclear halo into lumps in the transfected FHM cells. In addition, apoptotic features were found in the transfected FHM cells by overexpression of ORF3141 and ORF3141{Delta}sig proteins, respectively. Quantitative assays of mitochondrial membrane potential value, caspase-3 activity and apoptosis-related gene mRNA expression suggested that cell apoptosis was induced in the transfected FHM cells. In conclusion, the ORF3141 was a secreted protein of N. seriolae that targeted host cell mitochondria and induced apoptosis in FHM cells. This protein may participate in the cell apoptosis regulation and plays an important role in the pathogenesis of N. seriolae.\n\nAuthor summaryNocardia seriolae is the causative pathogen responsible for fish nocardiosis. This facultative intercellular bacterium, adapts to survive and colonize by evading intracellular killing after being engulfed with macrophages in the host. Despite considerable economic losses caused by N. seriolae in fish infection, the pathogenic mechanism and specific virulence factor of this bacterium remain ambiguous. In this study, the characteristic of ORF3141 protein function was investigated by subcellular localization and its possible contributions on the ability of N. seriolae to induce apoptosis in transfected fathead minnow (FHM) cells was investigated. Here, we confirmed that ORF3141 was a secreted protein that targeted host cell mitochondria and induced cell apoptosis in FHM cells. Interestingly, after deleting the signal peptide, ORF3141{Delta}sig protein was evenly distributed in the whole host cell and did not co-localize with the mitochondria which could also induce cell apoptosis. Thus, the N-terminate signal peptide played an important role in mitochondrial targeting, and the domain part without the signal peptide had a critical relationship with cell apoptosis. These results demonstrated that ORF3141 mays act as a potential virulence factor that induces apoptosis in fish cells. This protein is significant to elucidate the pathogenic mechanism of N. seriolae and this study mays provide beneficial insight to prevent and treat fish nocardiosis.

pathology↗

bioRxiv2018

An efficient and improved laboratory workflow and tetrapod database for larger scale eDNA studies

BackgroundThe use of environmental DNA, eDNA, for species detection via metabarcoding is growing rapidly. We present a co-designed lab workflow and bioinformatic pipeline to mitigate the two most important risks of eDNA: sample contamination and taxonomic mis-assignment. These risks arise from the need for PCR amplification to detect the trace amounts of DNA combined with the necessity of using short target regions due to DNA degradation. FindingsOur high-throughput workflow minimises these risks via a four-step strategy: (1) technical replication with two PCR replicates and two extraction replicates; (2) using multi-markers (12S, 16S, CytB); (3) a twin-tagging, two-step PCR protocol;(4) use of the probabilistic taxonomic assignment method PROTAX, which can account for incomplete reference databases. As annotation errors in the reference sequences can result in taxonomic mis-assignment, we supply a protocol for curating sequence datasets. For some taxonomic groups and some markers, curation resulted in over 50% of sequences being deleted from public reference databases, due to (1) limited overlap between our target amplicon and reference sequences; (2) mislabelling of reference sequences; (3) redundancy. Finally, we provide a bioinformatic pipeline to process amplicons and conduct PROTAX assignment and tested it on an invertebrate derived DNA (iDNA) dataset from 1532 leeches from Sabah, Malaysia. Twin-tagging allowed us to detect and exclude sequences with non-matching tags. The smallest DNA fragment (16S) amplified most frequently for all samples, but was less powerful for discriminating at species rank. Using a stringent and lax acceptance criteria we found 162 (stringent) and 190 (lax) vertebrate detections of 95 (stringent) and 109 (lax) leech samples. ConclusionsOur metabarcoding workflow should help research groups increase the robustness of their results and therefore facilitate wider usage of e/iDNA, which is turning into a valuable source of ecological and conservation information on tetrapods.

molecular biology↗

bioRxiv2018

Metagenomic screening of global microbiomes identifies pathogen-enriched environments

BackgroundHuman pathogens are widespread in the environment, and examination of pathogen-enriched environments in a rapid and high-throughput fashion is important for development of pathogen-risk precautionary measures.\n\nMethodsIn this study, a Local BLASTP procedure for metagenomic screening of pathogens in the environment was developed using a toxin-centered database. A total of 27 microbiomes derived from ocean water, freshwater, soil, feces, and wastewater were screened using the Local BLASTP procedure. Bioinformatic analysis and Canonical Correspondence Analysis were conducted to examine whether the toxins included in the database were taxonomically associated.\n\nResultsThe specificity of the Local BLASTP method was tested with known and unknown toxin sequences. Bioinformatic analysis indicated that most toxins were phylum-specific but not genus-specific. Canonical Correspondence Analysis implied that almost all of the toxins were associated with the phyla of Proteobacteria, Nitrospirae and Firmicutes. Local BLASTP screening of the global microbiomes showed that pore-forming RTX toxin and adenylate cyclase Cya were most prevalent globally in terms of relative abundance, while polluted water and feces samples were the most pathogen-enriched.\n\nConclusionsA Local BLASTP procedure was established for rapid detection of toxins in environmental samples. Screening of global microbiomes in this study provided a quantitative estimate of the most prevalent toxins and most pathogen-enriched environment.

ecology↗

bioRxiv2018

artMAP: a user-friendly tool for mapping EMS-induced mutations in Arabidopsis

Mapping-by-sequencing is a rapid method for identifying both natural as well as induced variations in the genome. However, it requires extensive bioinformatics expertise along with the computational infrastructure to analyze the sequencing data and these requirements have limited its widespread adoption. In the current study, we develop an easy to use tool, artMAP, to discover ethyl methanesulfonate (EMS) induced mutations in the Arabidopsis genome. The artMAP pipeline consists of well-established tools including TrimGalore, BWA, BEDTools, SAMtools, and SnpEff which were integrated in a Docker container. artMAP provides a graphical user interface and can be run on a regular laptop and desktop, thereby limiting the bioinformatics expertise required. artMAP can process input sequencing files generated from single or paired-end sequencing. The results of the analysis are presented in interactive graphs which display the annotation details of each mutation. Due to its ease of use, artMAP made the identification of EMS-induced mutations in Arabidopsis possible with only a few mouse click. The source code of artMAP is available on Github (https://github.com/RihaLab/artMAP).

plant biology↗

bioRxiv2018

Double-digest RAD-sequencing: do wet and dry protocol parameters impact biological results?

O_LINext-generation sequencing technologies have opened a new era of research in genomics. Among these, restriction enzyme-based techniques such as restriction-site associated DNA sequencing (RADseq) or double-digest RAD-sequencing (ddRADseq) are now widely used in many population genomics fields. From DNA sampling to SNP calling, both wet and dry protocols have been discussed in the literature to identify key parameters for an optimal loci reconstruction.\nC_LIO_LIThe impact of these parameters on downstream analyses and biological results drawn from RADseq or ddRADseq data has however not been fully explored yet. In this study, we tackled this issue by investigating the effects of ddRADseq laboratory (i.e. wet protocol) and bioinformatics (i.e. dry protocol) settings on loci reconstruction and inferred biological signal at two evolutionary scale using two systems: a complex of butterfly species (Coenonympha sp.) and populations of Common beech (Fagus sylvatica).\nC_LIO_LIResults suggest an impact of wet protocol parameters (DNA quantity, number of PCR cycles during library preparation) on the number of recovered reads and SNPs, the number of unique alleles and individual heterozygosity. We also found that bioinformatic settings (i.e. clustering and minimum coverage thresholds) impact loci reconstruction (e.g. number of loci, mean coverage) and SNP calling (e.g. number of SNPs, heterozygosity). We however do not detect an impact of parameter settings on three types of analysis performed with ddRADseq data: measure of genetic differentiation, estimation of individual admixture, and demographic inferences. In addition, our work demonstrates the high reproducibility and low rate of genotyping inconsistencies of the ddRADseq protocol.\nC_LIO_LIThus, our study highlights the impact of wet parameters on ddRADseq protocol with strong consequences on experimental success and biological conclusions. Dry parameters affects loci reconstruction and descriptive statistics but not biological conclusion for the two studied systems. Overall, this study illustrates, with others, the relevance of ddRADseq for population and evolutionary genomics at the inter- or intraspecific scales.\nC_LI

molecular biology↗

bioRxiv2018

SKA: Split Kmer Analysis Toolkit for Bacterial Genomic Epidemiology

Genome sequencing is revolutionising infectious disease epidemiology, providing a huge step forward in sensitivity and specificity over more traditional molecular typing techniques. However, the complexity of genome data often means that its analysis and interpretation requires high-performance compute infrastructure and dedicated bioinformatics support. Furthermore, current methods have limitations that can differ between analyses and are often opaque to the user, and their reliance on multiple external dependencies makes reproducibility difficult. Here I introduce SKA, a toolkit for analysis of genome sequence data from closely-related, small, haploid genomes. SKA uses split kmers to rapidly identify variation between genome sequences, making it possible to analyse hundreds of genomes on a standard home computer. Tests on publicly available simulated and real-life data show that SKA is both faster and more efficient than the gold standard methods used today while retaining similar levels of accuracy for epidemiological purposes. SKA can take raw read data or genome assemblies as input and calculate pairwise distances, create single linkage clusters and align genomes to a reference genome or using a reference-free approach. SKA requires few decisions to be made by the user, which, along with its computational efficiency, allows genome analysis to become accessible to those with only basic bioinformatics training. The limitations of SKA are also far more transparent than for current approaches, and future improvements to mitigate these limitations are possible. Overall, SKA is a powerful addition to the armoury of the genomic epidemiologist. SKA source code is available from Github (https://github.com/simonrharris/SKA).

genomics↗

bioRxiv2018

Definitive demonstration by synthesis of genome annotation completeness

Bacteriophage oX174 was the first DNA genome to be sequenced. The genome is well studied by classical methods and is known to encode 11 essential genes. At least 23 closely-related Bullavirinae genome sequences are now available. We identified 315 potential open reading frames (ORFs) within the genome via bioinformatic analysis, and a subset of 82 highly-conserved ORFs that have no known gene products or functions. Using genome scale design and synthesis we made a mutant genome in which all 11 essential genes are simultaneously disrupted, leaving intact only the 82 conserved-but-cryptic ORFs. The resulting genome is not viable, as expected. Cell-free gene expression followed by mass spectrometry revealed only a single peptide expressed from both the cryptic-ORF and wild-type genomes, suggesting a potential new gene. A second synthetic genome in which 71 conserved cryptic ORFs were simultaneously disrupted is viable but with ~50% reduced fitness relative to the wild type. However, rather than finding any new genes, repeated evolutionary adaptation revealed a single point mutation modulating translation of gene H, a known essential gene, that fully suppressed the fitness defect. Taken together, we conclude that the annotation of ORFs for the oX174 genome is formally complete. Sequencing and bioinformatics followed by synthesis-enabled reverse genomics, proteomics, and evolutionary adaptation can definitely establish the sufficiency and completeness of natural genome annotations.

synthetic biology↗

bioRxiv2018

Comparative Transcriptome and Methylome Analysis in Human Skeletal Muscle Anabolism, Hypertrophy and Epigenetic Memory.

Transcriptome wide changes in human skeletal muscle after acute (anabolic) and chronic resistance exercise (RE) induced hypertrophy have been extensively determined in the literature. We have also recently undertaken DNA methylome analysis (850,000 + CpG sites) in human skeletal muscle after acute and chronic RE, detaining and retraining, where we identified an association between DNA methylation in an epigenetic memory of exercise induced skeletal muscle hypertrophy. However, it is currently unknown as to whether all the genes identified in the transcriptome studies to date are also epigenetically regulated at the DNA level after acute, chronic or repeated RE exposure. We therefore aimed to undertake large scale bioinformatical analysis by pooling the publicly available transcriptome data after acute (110 samples) and chronic RE (181 samples) and comparing these large data sets with our genome-wide DNA methylation analysis in human skeletal muscle after acute and chronic RE, detraining and retraining. Indeed, after acute RE we identified 866 up- and 936 down-regulated genes at the expression level, with 270 (out of the 866 up- regulated) identified as being hypomethylated, and 216 (out of 936 downregulated) as hypermethylated. After chronic RE we identified 2,018 up- and 430 down-regulated genes with 592 (out of 2,018 upregulated) identified as being hypomethylated and 98 (out of 430 genes downregulated) as hypermethylated. After KEGG pathway analysis, genes associated with cancer pathways were significantly enriched in both bioinformatic analysis of the pooled transcriptome and methylome data after both acute and chronic RE. This resulted in 23 (out of 69) and 28 (out of 49) upregulated and hypomethylated and 12 (out of 37) and 2 (out of 4) downregulated and hypermethylated cancer genes following acute and chronic RE respectively. Within skeletal muscle tissue, these cancer genes predominant functions were associated with matrix/ actin structure and remodelling, mechano-transduction (including PTK2/Focal Adhesion Kinase and Phospholipase D-following chronic RE only), TGF-beta signalling and protein synthesis (GSK3B after acute RE only). Interestingly, 51 genes were also identified to be up/downregulated in both the acute and chronic RE pooled transcriptome analysis as well as significantly hypo/hypermethylated after acute RE, chronic RE, detraining and retraining. Five genes; FLNB, MYH9, SRGAP1, SRGN, ZMIZ1 demonstrated increased gene expression in the acute and chronic RE transcriptome and also demonstrated hypomethylation in these conditions. Importantly, these 5 genes demonstrated retained hypomethylation even during detraining (following training induced hypertrophy) when exercise was ceased and lean mass returned to baseline (pre-training) levels, identifying them as potential epigenetic memory genes. Importantly, for the first time across the transcriptome and epigenome combined, this study identifies novel differentially methylated genes associated with human skeletal muscle anabolism, hypertrophy and epigenetic memory.

physiology↗

bioRxiv2018

Reduced mitochondrial lipid oxidation leads to fat accumulation in myosteatosis

Myosteatosis is the pathological accumulation of lipid that can occur in conjunction with atrophy and fibrosis following skeletal muscle injury. Little is known about the mechanisms by which lipid accumulates in myosteatosis, but many clinical studies have demonstrated the degree of lipid infiltration negatively correlates with muscle function and regeneration. Our objective was to determine the pathological changes that result in lipid accumulation in injured muscle fibers. We used a rat model of rotator cuff injury in this study, as the rotator cuff muscle group is particularly prone to the development of myosteatosis after injury. Muscles were collected from uninjured controls, or 10, 30, or 60 days after injury, and analyzed using a combination of muscle fiber contractility assessments, RNA sequencing, and undirected metabolomics, lipidomics and proteomics, along with bioinformatics techniques, to identify potential pathways and cellular processes that are dysregulated after rotator cuff tear. Bioinformatics analyses indicated that mitochondrial function was likely disrupted after injury. Based on these findings, and given the role that mitochondria play in lipid metabolism, we then performed targeted biochemical and imaging studies and determined that mitochondrial dysfunction and reduced fatty acid oxidation likely leads to the accumulation of lipid in myosteatosis.

physiology↗