Construction of a virtual Mycobacterium tuberculosis consensus genome and its application to data from a next generation sequencer

Background

Although Mycobacterium tuberculosis isolates are consisted of several different lineages and the epidemiology analyses are usually assessed relative to a particular reference genome, M. tuberculosis H37Rv, which might introduce some biased results. Those analyses are essentially based genome sequence information of M. tuberculosis and could be performed in sillico in theory, with whole genome sequence (WGS) data available in the databases and obtained by next generation sequencers (NGSs). As an approach to establish higher resolution methods for such analyses, whole genome sequences of the M. tuberculosis complexes (MTBCs) strains available on databases were aligned to construct virtual reference genome sequences called the consensus sequence (CS), and evaluated its feasibility in in sillico epidemiological analyses.

Results

The consensus sequence (CS) was successfully constructed and utilized to perform phylogenetic analysis, evaluation of read mapping efficacy, which is crucial for detecting single nucleotide polymorphisms (SNPs), and various MTBC typing methods virtually including spoligotyping, VNTR, Long sequence polymorphism and Beijing typing. SNPs detected based on CS, in comparison with H37Rv, were utilized in concatemer-based phylogenetic analysis to determine their reliability relative to a phylogenetic tree based on whole genome alignment as the gold standard. Statistical comparison of phylogenic trees based on CS with that of H37Rv indicated the former showed always better results that that of later. SNP detection and concatenation with CS was advantageous because the frequency of crucial SNPs distinguishing among strain lineages was higher than those of H37Rv. The number of SNPs detected was lower with the consensus than with the H37Rv sequence, resulting in a significant reduction in computational time. Performance of each virtual typing was satisfactory and accorded with those published when those are available.

Conclusions

These results indicated that virtual CS constructed from genome sequence data is an ideal approach as a reference for MTBC studies.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1368-9) contains supplementary material, which is available to authorized users.     

Complete Nucleotide Sequence and Genetic Organization of Aichi Virus, a Distinct Member of the Picornaviridae Associated with Acute Gastroenteritis in Humans

The complete nucleotide sequence of a novel enteric virus, Aichi virus, associated with nonbacterial acute gastroenteritis in humans was determined. The Aichi virus genome proved to be a single-stranded positive-sense RNA molecule with 8,251 bases excluding a poly(A) tail; it contains a large open reading frame with 7,302 nucleotides that encodes a potential polyprotein precursor of 2,433 amino acids. The genome contains a 5′ nontranslated region (NTR) with 712 bases and a 3′ NTR with 240 bases followed by a poly(A) tail. The structure of the genome, VPg–5′ NTR–leader protein–structural proteins–nonstructural proteins–3′ NTR–poly(A), was found to be typical of a picornavirus. The VP0-VP3 and VP3-VP1 cleavage sites were determined to be Q-H and Q-T, respectively, by N-terminal amino acid sequence analyses using purified virion proteins. Possible cleavage sites, Q-G, Q-A, and Q-S, which cleave P2 and P3 polyproteins were found to be similar to those of picornaviruses. A dendrogram based on 3D^pol proteins indicated that Aichi virus is genetically distinct from the known six genera of picornaviruses including entero-, rhino-, cardio-, aphtho-, and hepatovirus and echovirus 22. Considering this together with other properties of the virus (T. Yamashita, S. Kobayashi, K. Sakae, S. Nakata, S. Chiba, Y. Ishihara, and S. Isomura, J. Infect. Dis. 164:954–957, 1991), we propose that Aichi virus be regarded as a new genus of the family Picornaviridae.     

The HNF-1 target collectrin controls insulin exocytosis by SNARE complex formation

Defective glucose-stimulated insulin secretion is the main cause of hyperglycemia in type 2 diabetes mellitus. Mutations in HNF-1 cause a monogenic form of type 2 diabetes, maturity-onset diabetes of the young (MODY), characterized by impaired insulin secretion. Here we report that collectrin, a recently cloned kidney-specific gene of unknown function, is a target of HNF-1 in pancreatic β cells. Expression of collectrin was decreased in the islets of HNF-1 (−/−) mice, but was increased in obese hyperglycemic mice. Overexpression of collectrin in rat insulinoma INS-1 cells or in the β cells of transgenic mice enhanced glucose-stimulated insulin exocytosis, without affecting Ca²⁺ influx. Conversely, suppression of collectrin attenuated insulin secretion. Collectrin bound to SNARE complexes by interacting with snapin, a SNAP-25 binding protein, and facilitated SNARE complex formation. Therefore, collectrin is a regulator of SNARE complex function, which thereby controls insulin exocytosis.     

A Growth Factor for Malo-lactic Fermentation Bacteria

A growth factor (TJF) for a malo-lactic fermentation bacterium has been isolated from tomato juice, and found to be a β-glucoside. The NMR spectra of TJF and its acetate revealed that the glucosyl residue linked to the hydroxyl group at C-2′ or C-4′ of d- or l-pantothenic acid moiety. Then, 2′-O-(β-d-glucopyranosyl)-dl-pantothenic acid (I), 4′-O-(β-d-glucopyranosyl)-dl-pantothenic acid (II) and 4′-O-(β-d-glucopyranosyl)-d(R)-pantothenic acid (II-a) were synthesized, and Il-a and 4′-O-(β-d-glucopyranosyl)-l-pantothenic acid (II-b) were obtained by the optical resolution of the acetate of II. Among the above compounds, II-a was identical with natural TJF regarding to the biological activity, NMR and ORD spectra, and thin-layer chromatography.     

The 135-kDa actin-bundling protein from lily pollen tubes arranges F-actin into bundles with uniform polarity

 A plant 135-kDa actin-bundling protein (P-135-ABP) isolated from pollen tubes of Lilium longiflorum (Thunb.) binds stoichiometrically to F-actin filaments and bundles them in vitro (E. Yokota et al., 1998, Plant Physiol. 116: 1421–1429). To further understand the mechanism of actin-filament bundle formation by P-135-ABP, the polarity of each F-actin filament in bundles was examined using myosin subfragment 1 (S-1). Dissociation of F-actin filaments from bundles organized by P-135-ABP was induced by S-1. However, F-actin filaments that remained in a bundle and decorated by S-1 showed uniform polarity. These results indicate that P-135-ABP arranges F-actin filaments into bundles with uniform polarity and consequently plays a key role in the orientation of cytoplasmic streaming in pollen tubes. Received: 23 February 1999 / Accepted: 22 April 1999     

A Novel Cell-Sheet Technology That Achieves Durable Factor VIII Delivery in a Mouse Model of Hemophilia A

Gene- or cell-based therapies aimed at creating delivery systems for coagulation factor VIII (FVIII) protein have emerged as promising options for hemophilia A treatment. However, several issues remain to be addressed regarding the efficacies and adverse events of these new classes of therapies. To improve an existing cell-based therapy involving the subcutaneous transplantation of FVIII-transduced blood outgrowth endothelial cells (BOECs), we employed a novel cell-sheet technology that allows individual dispersed cells to form a thin and contiguous monolayer without traditional bioabsorbable scaffold matrices. Compared to the traditional methodology, our cell-sheet approach resulted in longer-term and 3–5-fold higher expression of FVIII (up to 11% of normal) in recipient hemophilia A mice that lacked a FVIII humoral immune response due to transient immunosuppression with cyclophosphamide. Histological studies revealed that the transplanted BOEC sheets were structured as flat clusters, supporting the long-term expression of therapeutic FVIII in plasma from an ectopic subcutaneous space. Our novel tissue-engineering approach using genetically modified BOEC sheets could aid in development of cell-based therapy that will allow safe and effective in vivo delivery of functional FVIII protein in patients with hemophilia A.