Basic Local Alignment Search Tool Search Research Articles

Occurrence of Praxelis clematidea Witches'-Broom Disease Association with 16SrI Group 'Candidatus Phytoplasma asteris' in Hainan Island of China.

Praxelis clematidea is an invasive herbaceous plant belonging to Asteraceae family. From August to November 2020, the plants showing severe witches'-broom symptoms were found in farms and roadsides from Ding'an of Hainan Province, a tropical island of China. The disease symptoms were suggestive of phytoplasma infection. For pathogen detection, P. clematidea samples consisting of six symptomatic and three asymptomatic plants were collected from the farms and roadsites of Ding'an with 40 % incidence by conducting surveys and statistics. Total nucleic acids were extracted using 0.10 g of fresh leaf tissues of the plant through CTAB DNA extraction method. Conserved gene sequences of 16S rRNA and secA genes from phytoplasma were amplified by direct PCR using primer pairs of R16mF2/R16mR1 and secAfor1/secArev3, respectively. R16mF2/R16mR1 PCR amplicons were obtained for all symptomatic samples but not from the symptomless plants. The amplicons were purified and sequenced by Biotechnology (Shanghai) Co., Ltd. (Guangzhou, China). Sequences of 16S rRNA gene (1323 bp) and secA (732 bp) were obtained and all the gene sequences were identical, designated as PcWB (Praxelis clematidea witches'-broom)-hnda. Representative sequencs were deposited in Genbank with accession numbers of PP098736 (16S rDNA) and PP072216 (secA). Nucleotide BLAST (Basic Local Alignment Search Tool) search based on 16S rRNA gene sequences indicated that PcWB-hnda had 100% sequence identity (1323/1323) with 'Candidatus Phytoplasma asteris'-related strains belonging to 16SrI group like Waltheria indica virescence phytoplasma (MW353909) and Capsicum annuum yellow crinkle phytoplasma (MT760793); had 99.62 % sequence identity (1321/1326) with the phytoplasma strains of 16SrI group such as Oenothera phytoplasma (M30790). RFLP (Restriction Fragment Length Polymorphism) pattern derived from 16Sr RNA gene sequences by iPhyClassifier showed identical (similarity coefficient=1.00) to the reference pattern of 16SrI-B subgroup (GenBank accession number: AP006628). The results obtained demonstrate that the phytoplasma strain PcWB-hnda under study is a member of 16SrI-B subgroup. A BLAST search based on secA gene sequences indicated that PcWB-hnda shares 100% sequence identity (732/732 bp) with Pericampylus glaucus witches'-broom phytoplasma (MT875200), 99% sequence identify (728/732 bp) with onion yellows phytoplasma OY-M(AP006628), and 99% sequence identify (729/732 bp) with rapeseed phyllody phytoplasma isolate RP166 (CP055264), among other phytoplasma strains that belong to 16SrI group. Previous studies demonstrated that P. clematidea can be infected by phytoplasmas affiliate to the 16SrII group (GenBank accession number: KY568717 and EF061924) in Hainan Island of China. To our knowledge, this is the first report of a natural infection of P. clematidea by a group 16SrI phytoplasma in the Island of China. 16SrI group can infect agronomic important species such as areca palm in the island and P. clematidea can be a reservoir of 16SrI phytoplasmas. Therefore, it is necessary to search of potential vectors of the pathogens, which would contribute to epidemiological monitoring and prevention of the related diseases.

ElasticBLAST: accelerating sequence search via cloud computing

BackgroundBiomedical researchers use alignments produced by BLAST (Basic Local Alignment Search Tool) to categorize their query sequences. Producing such alignments is an essential bioinformatics task that is well suited for the cloud. The cloud can perform many calculations quickly as well as store and access large volumes of data. Bioinformaticians can also use it to collaborate with other researchers, sharing their results, datasets and even their pipelines on a common platform.ResultsWe present ElasticBLAST, a cloud native application to perform BLAST alignments in the cloud. ElasticBLAST can handle anywhere from a few to many thousands of queries and run the searches on thousands of virtual CPUs (if desired), deleting resources when it is done. It uses cloud native tools for orchestration and can request discounted instances, lowering cloud costs for users. It is supported on Amazon Web Services and Google Cloud Platform. It can search BLAST databases that are user provided or from the National Center for Biotechnology Information.ConclusionWe show that ElasticBLAST is a useful application that can efficiently perform BLAST searches for the user in the cloud, demonstrating that with two examples. At the same time, it hides much of the complexity of working in the cloud, lowering the threshold to move work to the cloud.

DOP50 Impact of Inflammatory Bowel Disease associated dysbiosis on bacterial quorum sensing mediated by Acyl-homoserine Lactone in human gut microbiota

Abstract Background Intestinal dysbiosis is a key feature in the pathogenesis of inflammatory bowel diseases (IBD). Bacterial quorum sensing mediated by acyl-homoserine lactones (AHL) might play a role in the dialogue between the gut microbiota and the host. The main objective of our study was to investigate the presence and expression of AHL synthase and receptor genes in the human gut ecosystem during IBD. Methods To confirm the presence of AHL in the gut, mass spectrometric detection was performed on stool samples from IBD patients and non-IBD subjects. Then, by an in silico approach, we exploited the open access database: Inflammatory Bowel Disease Multi’omics Database, an American cohort with bacterial metagenomes and metatranscriptomes data of stool samples from non-IBD and IBD subjects. To characterise gut dysbiosis, the most discriminating bacterial species between non-IBD and IBD patients were identified by multivariate analysis and allowed us to define two groups (dysbiosis/non-dysbiosis). The search for AHL synthase (luxI) and receptor (luxR) known homolog genes, was performed using Basic Local Alignment Search Tool (BLAST) from previously assembled gene files (presence/absence) as well as raw data sequencing files (relative abundance and expression). Results Mass spectrometry confirmed a higher concentration of AHL molecules in healthy subjects than in relapsing IBD. Regarding in silico analysis, 103 subjects were selected including 50 with Crohn’s disease (CD), 27 with ulcerative colitis (UC), and 26 non-IBD subjects. No luxI-like synthase genes were retrieved by BLAST searches. However, several homologs of receptor genes were identified: sdiA gene from Escherichia coli (7/103 patients) and luxR-like homologs from Bacteroides fragilis and Bacteroides dorei present in all patients. According to disease, only one luxR-like gene from Bacteroides dorei was under-expressed in IBD patients (p = 0.02) compared to non-IBD, especially in CD (p = 0.02) (Figure 1). In dysbiosis situation, one luxR receptor gene from Bacteroides fragilis appeared to be over-expressed (p = 0.04) compared to non-dysbiotic patients (Figure 2). Conclusion Through this computational approach, AHL-synthesising bacteria have not been found. However, the expression of quorum sensing receptor genes appears to be modulated by IBD-associated gut dysbiosis. The role of LuxR receptors, especially in Bacteroides species, should be investigated to understand its impact on gut microbiota (Figure 3). Targeting LuxR receptors of bacterial quorum sensing might represent a new approach to modulate the gut microbiota in IBD.

AlgPred 2.0: an improved method for predicting allergenic proteins and mapping of IgE epitopes.

AlgPred 2.0 is a web server developed for predicting allergenic proteins and allergenic regions in a protein. It is an updated version of AlgPred developed in 2006. The dataset used for training, testing and validation consists of 10075 allergens and 10075 non-allergens. In addition, 10451 experimentally validated immunoglobulin E (IgE) epitopes were used to identify antigenic regions in a protein. All models were trained on 80% of data called training dataset, and the performance of models was evaluated using 5-fold cross-validation technique. The performance of the final model trained on the training dataset was evaluated on 20% of data called validation dataset; no two proteins in any two sets have more than 40% similarity. First, a Basic Local Alignment Search Tool (BLAST) search has been performed against the dataset, and allergens were predicted based on the level of similarity with known allergens. Second, IgE epitopes obtained from the IEDB database were searched in the dataset to predict allergens based on their presence in a protein. Third, motif-based approaches like multiple EM for motif elicitation/motif alignment and search tool have been used to predict allergens. Fourth, allergen prediction models have been developed using a wide range of machine learning techniques. Finally, the ensemble approach has been used for predicting allergenic protein by combining prediction scores of different approaches. Our best model achieved maximum performance in terms of area under receiver operating characteristic curve 0.98 with Matthew's correlation coefficient 0.85 on the validation dataset. A web server AlgPred 2.0 has been developed that allows the prediction of allergens, mapping of IgE epitope, motif search and BLAST search (https://webs.iiitd.edu.in/raghava/algpred2/).

English

Environmental pollution is principally caused by human activities that usually result in the release of man–made pollutants such as biological, chemical and radioactive in such states as solid, liquid and gaseous substances into the biosphere via, land, water and air. Globally, the increasing rate of environmental contamination by ammunition/explosives resulting from their increasing deployments in the rising spites of military conflicts and training has certainly become a matter of great concern for every nation. The aim of this research was to molecularly characterize some isolated bacteria from an apparent munitions contaminated sites in Kachia Military Firing Range, Kaduna State. DNA from each isolated bacteria was extracted and 16s rRNA Gene amplified from each isolated bacteria DNA using thermo cycler. The amplified genes were run on Agarose gel plate and visualized. Amplified gene bands were sequenced and Basic Local Alignment Search Tool (BLAST). 16s rRNA gene sequences result aligned with BLAST search of NCBI databases that revealed the presence of Lysinibacillus pakistanensis, Eschericia coli, Achromobacter spanius, Achromobacter animicus, Escherichia fergusonii and Shigella sp. The results identified bacteria that were isolated from munitions contaminated sites and that could also be useful for their bioremediation potential against munitions contaminant. Key words: Environment, pollution, contamination, bacteria, bioremediation.

Alignment-free approaches for predicting novel Nuclear Mitochondrial Segments (NUMTs) in the human genome

The nuclear human genome harbors sequences of mitochondrial origin, indicating an ancestral transfer of DNA from the mitogenome. Several Nuclear Mitochondrial Segments (NUMTs) have been detected by alignment-based sequence similarity search, as implemented in the Basic Local Alignment Search Tool (BLAST). Identifying NUMTs is important for the comprehensive annotation and understanding of the human genome. Here we explore the possibility of detecting NUMTs in the human genome by alignment-free sequence similarity search, such as k-mers (k-tuples, k-grams, oligos of length k) distributions. We find that when k=6 or larger, the k-mer approach and BLAST search produce almost identical results, e.g., detect the same set of NUMTs longer than 3 kb. However, when k=5 or k=4, certain signals are only detected by the alignment-free approach, and these may indicate yet unrecognized, and potentially more ancestral NUMTs. We introduce a “Manhattan plot” style representation of NUMT predictions across the genome, which are calculated based on the reciprocal of the Jensen-Shannon divergence between the nuclear and mitochondrial k-mer frequencies. The further inspection of the k-mer-based NUMT predictions however shows that most of them contain long-terminal-repeat (LTR) annotations, whereas BLAST-based NUMT predictions do not. Thus, similarity of the mitogenome to LTR sequences is recognized, which we validate by finding the mitochondrial k-mer distribution closer to those for transposable sequences and specifically, close to some types of LTR.

Hypothetical Protein from Aspergillus niger contains Chromate Reductase Motifs

Chromate reductase is an enzyme that converts toxic and carcinogenic Cr6+ to non-carcinogenic and less soluble Cr3+. A hypothetical protein (ref.XP-001388504.1) from Aspergillus niger has been identified as chromate reductase using bioinformatics techniques. The techniques involved Clustal W alignment and Basic Local Alignment Search Tool (BLAST). Using Clustal W alignment the motifs VTPEYN and LKNAID motifs were identified to be common among the different chromate reductase homologues aligned. The same motifs reappeared when the hypothetical protein was aligned with other chromate reductase homologues. The BLAST search was able to also identify VTPEYN and LKNAID motifs on the A. niger genome indicating the likelihood of the hypothetical protein being chromate reductase. The homologue was recovered as putative chromate reductase gene encoded by an open reading frame of 2227 bp nucleotide sequence. These findings clearly demonstrate the hypothetical protein is likely to be chromate reductase.

CABRA: Cluster and Annotate Blast Results Algorithm.

BackgroundBasic local alignment search tool (BLAST) searches are frequently used to look for homologous sequences and to annotate a query protein, but the increasing size of protein databases makes it difficult to review all results from a similarity search.FindingsWe developed a web tool called Cluster and Annotate Blast Results Algorithm (CABRA), which enables a rapid BLAST search in a variety of updated reference proteomes, and provides a new way to functionally evaluate the results by the subsequent clustering of the hits and annotation of the clusters. The tool can be accessed from the following web-resource: http://cbdm-01.zdv.uni-mainz.de/~munoz/CABRA.ConclusionsCluster and Annotate Blast Results Algorithm simplifies the analysis of the results of a BLAST search by providing an overview of the result’s annotations organized in clusters that can be iteratively modified by the user.

A Case of Phaeohyphomycosis Caused by Exophiala lecanii-corni.

Dear Editor: Phaeohyphomycosis refers to subcutaneous and systemic fungal infections characterized by dematiaceous septate mycelial elements1. The most common agent of subcutaneous phaeohyphomycosis is Exophiala jeanselmei. Phaeohyphomycosis has been increasing in frequency because of popular immunosuppressive therapy and precise laboratory diagnosis2. To the best of our knowledge, there has been no report of subcutaneous phaeohyphomycosis due to Exophiala lecanii-corni in Korea. A 76-year-old man presented with ill-defined pruritic erythematous papules and patches on right forearm for 18 months. He had a history of swimming in the sea of Egyptian pharaoh island. After then, the skin lesion was developed and gradually increased in size. Before the travel, he had a history of trauma at the site of the lesion a week ago. He had undergone diabetes mellitus (DM) and hyperlipidemia for several years. On physical examination, skin lesion showed confluent flat-topped violaceous papules on ill-defined purpuric erythematous patches (Fig. 1A, B). Histopathological examination showed granuloma consisting of histiocytes and lymphocytes, fungal hyphae and spores in dermis (Fig. 2A~D). Fungal culture was done with potato dextrose agar (PDA) with corn meal agar, peptone and tween 80 for 14 days. Fungal culture showed dark brown to olivaceous black colored velvety colony (Fig. 2E). Microscopic examination using lactophenol cotton blue staining method revealed spine-like conidiophores and clusters of single celled conidia (Fig. 2F). We performed a direct sequencing analysis of the internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA) for the identification of causative fungus. The GenBank National Center for Biotechnology Information (NCBI) database using the basic local alignment search tool (BLAST) algorithm was used. The GenBank BLAST search revealed 100% (575/575 bp) homology with a previously sequenced E. lecanii-corni strain (GenBank accession number: {type:entrez-nucleotide,attrs:{text:AY857528.1,term_id:61676683,term_text:AY857528.1}}AY857528.1). The patient received oral terbinafine HCl 250 mg for 3 months and after then oral fluconazole 150 mg for 4 months. After treatment, the lesion was improved without recurrence (Fig. 1C, D). Fig. 1 (A) Confluent flat-topped violaceous papules on ill-defined purpuric erythematous patches, (B) close up view of the lesion. (C) Clinical improvement of skin lesion 11 months after treatment, (D) close up view of the lesion. Fig. 2 (A, B) Granuloma consisting of histiocytes and lymphocytes in dermis (HE A: ×40, B, ×400). (C, D) Fungal hyphae and spores in dermis (C: D-PAS, ×400, D: GMS, ×400). (E) Dark brown to olivaceous black-colored velvety ... Phaeohyphomycosis, named by Ajello et al.3 in 1974, was first defined. Phaeohyphomycosis refers to Dematiaceae infection, varying from subcutaneous mycosis to deep organ infections. Unlike chromoblastomycosis, phaeohyphomycosis shows black yeast-like cells, pseudomycelium-like fungal sections, mycelia, or their complex in tissues rather than sclerotic bodies or granulation4. The subcutaneous type frequently occurs in the fingers, wrists, knees, and ankles, which can be easily exposed to injury and wound infection5. It needs to be differentiated from fibroma, lipoma, and steatocystoma. Phaeohyphomycosis is increasing in immunocompromised patients such as solid organ transplant recipients, hemato-oncological patients, and patients on chronic exogenous steroid therapy2. The diagnosis of phaeohyphomycosis can be made by past history, clinical features, fungal culture, microscopic examination of fungal elements, histopathological examination, polymerase chain reaction and rRNA ITS sequence analyses. Our case was diagnosed as phaeohyphomycosis using all of the above methods. In the present case, E. lecanii-corni, which has not yet been reported in Korea, was identified. Among the Exophiala species, E. castellanii, E. jeanselmei, E. moniliae, E. pisciphila, E. salmonis, and E. spinifera are common. Here we report a rare case of phaeohyphomycosis induced by E. lecanii-corni.

A comparison of virus genome sequences with their host silkworm, Bombyx mori

With the recent availability of the genomes of many viruses and the silkworm, Bombyx mori, as well as a variety of Basic Local Alignment Search Tool (BLAST) programs, a new opportunity to gain insight into the interaction of viruses with the silkworm is possible. This study aims to determine the possible existence of sequence identities between the genomes of viruses and the silkworm and attempts to explain this phenomenon. BLAST searches of the genomes of viruses against the silkworm genome were performed using the resources of the National Center for Biotechnology Information. All studied viruses contained variable numbers of short regions with sequence identity to the genome of the silkworm. The short regions of sequence identity in the genome of the silkworm may be derived from the genomes of viruses in the long history of silkworm-virus interaction. This study is the first to compare these genomes, and may contribute to research on the interaction between viruses and the silkworm.

English

Following advances in DNA and protein sequencing, the application of computational approaches in analysing biological data has become a very important aspect of biology. Evaluating similarities between biological sequences is crucial to our understanding of evolutionary biology, and this can be achieved by basic local alignment search tool (BLAST) and fast alignment (FASTA). BLAST and FASTA have become fundamental tools of biology and it is essential to know how they operate, the task they can accomplish and how to accurately interpret their output. This paper provides an analysis of BLAST and FASTA in sequence analysis. Both BLAST and FASTA algorithms are appropriate for determining highly similar sequences. However, BLAST appears to be faster and also more accurate than FASTA. Both BLAST and FASTA are limited in sensitivity and may not be able to capture highly divergent sequences in some cases. Consequently, evolutionarily diverse members of a family of proteins may be missed out in a BLAST or FASTA search. Key words: Bioinformatics, basic local alignment search tool (BLAST), fast alignment (FASTA), sequence alignment, prokaryotes.

A Small-Group Activity Introducing the Use and Interpretation of BLAST

As biological sequence data are generated at an ever increasing rate, the role of bioinformatics in biological research also grows. Students must be trained to complete and interpret bioinformatic searches to enable them to effectively utilize the trove of sequence data available. A key bioinformatic tool for sequence comparison and genome database searching is BLAST (Basic Local Alignment Search Tool). BLAST identifies sequences in a database that are similar to the entered query sequence, and ranks them based on the length and quality of the alignment. Our goal was to introduce sophomore and junior level undergraduate students to the basic functions and uses of BLAST with a small group activity lasting a single class period. The activity provides students an opportunity to perform a BLAST search, interpret the data output, and use the data to make inferences about bacterial cell envelope structure. The activity consists of two parts. Part 1 is a handout to be completed prior to class, complete with video tutorial, that reviews cell envelope structure, introduces key terms, and allows students to familiarize themselves with the mechanics of a BLAST search. Part 2 consists of a hands-on, web-based small group activity to be completed during the class period. Evaluation of the activity through student performance assessments suggests that students who complete the activity can better interpret the BLAST output parameters % query coverage and % max identity. While the topic of the activity is bacterial cell wall structure, it could be adapted to address other biological concepts.

BioSpotlight

BioTechniquesVol. 53, No. 5 BioSpotlightOpen AccessBioSpotlightPatrick Lo & Kristie NyboPatrick LoSearch for more papers by this author & Kristie NyboSearch for more papers by this authorPublished Online:3 Apr 2018https://doi.org/10.2144/000113945AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInReddit Titer countsPhage display enables researchers to identify interactions of protein sequences fused to a phage coat protein expressed on the viral surface. Displayed sequences can be selected based on their desired binding properties, following which the associated virus is propagated in a bacterial host for isolation and purification. It is common to repeat the selection steps three or more times for enrichment, with the phage concentration determined after each propagation step. Phage concentration is commonly calculated using the plating method, where Escherichia coli are infected with phage, mixed in soft agar, plated, and the resulting plaques or colonies counted. Although this is an effective means of quantification, the procedure is lengthy, requiring an overnight incubation period. UV photometry can be used as a quicker approach to assess phage concentrations, but this method requires complete removal of impurities that absorb UV light and adversely affect readings. In this issue of BioTechniques, Lehmusvuori et al. from the University of Turku, Finland describe a new approach for quantifying bacteriophage using lanthanide fluorescence probes, a technology previously developed by the authors for other applications, and demonstrate its effectiveness using the M13 phage display system. The method requires two non-fluorescent oligonucleotide probes, one carrying a lanthanide ion and the other an antenna probe. When the probes hybridize to adjacent positions on the chloramphenicol acetyl-transferase gene, a selectable marker on the M13 phagemid single stranded DNA, the lanthanide ion and antenna probe form a highly fluorescent lanthanide chelate complex that can be measured as an indication of the concentration of ssDNA in solution. After only four hours in culture, phage could be detected in supernatant, demonstrating a significant time savings for this important step in phage display technology compared to the plating method. Measurements were highly reliable, and even more accurate than phage counts obtained using absorbance measurements. Although demonstrated with the M13 phage display system, this methodology should be applicable to quantification of other ssDNA phage species.Diagram of the complementation probe-based bacteriophage quantification assay detailed in Lehmusvuori, et al.See “Homogenous M13 bacteriophage quantification assay using switchable lanthanide fluorescence probes”.Having a BLAST (on your own)The first step in identifying any novel DNA or protein sequence is to perform a sequence similarity search against databases of known sequences to establish identity or find sequence homologs. The principal algorithm used for such similarity searches is the Basic Local Alignment Search Tool (BLAST), which is conveniently done online using the National Center for Biotechnology Information (NCBI) server, in conjunction with various publicly available databases such as GenBank and UniProt. Under certain circumstances, however, researchers may prefer to carry out offline BLAST searches on a local computer against their own databases of proprietary or unpublished sequences. While NCBI provides stand-alone, command line versions of BLAST that can be freely downloaded and installed on a personal computer, these are not particularly user-friendly, especially for biological researchers lacking computer expertise. There are two graphical front ends with extensive features and options for the BLAST command line software currently available, but these are restricted to Unix-based systems. Windows users unfortunately are restricted to a pair of graphical user interfaces (GUIs) that suffer from several major limitations. In this issue, P. Santiago-Sotelo and J.H. Ramirez-Prado at the Centro de Investigación Científica de Yucatán (Mexico) describe prfectBLAST, an operating system-independent GUI for the NCBI stand-alone suite of the new BLAST+ command line executables. prfectBLAST is free, open-source, completely self-contained, and coded in Java version 6, allowing it to be executed on multiple platforms (including Windows, Mac OS X, and Linux). In addition, the software does not require installation or any dependencies, so it can be run from an external flash or hard drive if desired. Its graphical layout and choice of options was designed to mirror the NCBI web interface for BLAST+ searches, and all BLAST+ parameters can be easily changed through the GUI. Searches using any of the BLAST+ programs are carried out against locally stored, user-customized databases and can be run in the computer background; several searches can be carried out concurrently. prfectBLAST is ideally suited for novice and casual users, yet flexible enough to allow experts to fully utilize all the advanced options available for BLAST+.See “prfectBLAST: a platform independent portable front end for the command terminal BLAST+ stand-alone suite”.FiguresReferencesRelatedDetails Vol. 53, No. 5 Follow us on social media for the latest updates Metrics History Published online 3 April 2018 Published in print November 2012 Information© 2012 Author(s)PDF download

Investigation of Borrelia burgdorferi genotypes in Australia obtained from erythema migrans tissue

BackgroundLyme disease (LD) is an emerging infectious disease in Australia. There has been controversy regarding endemic lyme disease in the country for over 20 years. Borrelia burgdorferi sensu stricto (Bbss) and sensu lato (Bbsl) are closely related spirochetal species that are the causative agents of LD in humans. Clinical transmission of this tick-borne disease is marked by a characteristic rash known as erythema migrans (EM). This study employed molecular techniques to demonstrate the spirochetal agent of Lyme disease isolated from EM biopsies of patients in Australia and then investigate their genetic diversity.MethodsFour patients who presented to the author’s practice over a one-year period from mid 2010 to mid 2011 returned positive results on central tissue biopsy of EM lesions using polymerase chain reaction (PCR) analysis. The findings were confirmed by DNA sequencing, and basic local alignment search tool (BLAST) analysis was then used to genetically characterize the causative organisms.ResultsThree isolates were identified as Bbss that lay genotypically between strains B31 and ZS7 and were then characterized as strain 64b. One of the three isolates though may have similarity to B. bissettii a Bbsl. The fourth isolate was more appropriately placed in the sensu lato group and appeared to be similar, but not identical to, a B. valaisiana-type isolate. In this study, a central biopsy taken within 6 days of infection was used instead of conventional sampling at the leading edge, and the merits of this are discussed.ConclusionThese patients acquired infection in Australia, further proving endemic LD on the continent. Central biopsy site of EM is a useful tool for PCR evaluation. BLAST searches suggest a genetic diversity of B. burgdorferi, which has implications concerning the diagnosis, clinical severity, and testing of LD in Australia.

Search for an aetiological virus candidate in chronic lymphocytic leukaemia by extensive transcriptome analysis

SummaryAs an approach to determining the aetiology of chronic lymphocytic leukaemia (CLL), we searched for a virus expressed in human CLL B‐cells by combining high‐throughput sequencing and digital subtraction. Pooled B‐cell mRNA transcriptomes from five CLL patients and five healthy donors were sequenced with 454 Life Sciences technology. Human reads were excluded by BLAST (Basic Local Alignment Search Tool) and BLAT (BLAST‐like alignment tool) searches. Remaining reads were screened with BLAST against viral databases. Purified B‐cells from two CLL patients, with and without stimulation by phorbol‐esters, were sequenced using Illumina technology to achieve depth of sequencing. Burrows‐Wheeler Aligner mapping and BLAST searches were used for the Illumina data. Pyrosequencing resulted in about 400 000 reads per sample. No viral candidate could be found. Illumina single‐end sequencing for 115 cycles yielded an average of 26 ± 2·5 million filtered reads per sample, of which 2·2 ± 0·6 million remained unmapped to human references. BLAST searches of these reads against viral and human databases assigned nine reads to an Epstein‐Barr virus origin, in one sample following phorbol‐ester stimulation. Other reads showing a putative viral origin were dismissed after further analysis. Despite an in‐depth analysis of the CLL transcriptome reaching more than 100 million sequences, we have not found evidence for a putative viral candidate in CLL.

Proteomic Profiling and Protein Identification by MALDI-TOF Mass Spectrometry in Unsequenced Parasitic Nematodes

Lack of genomic sequence data and the relatively high cost of tandem mass spectrometry have hampered proteomic investigations into helminths, such as resolving the mechanism underpinning globally reported anthelmintic resistance. Whilst detailed mechanisms of resistance remain unknown for the majority of drug-parasite interactions, gene mutations and changes in gene and protein expression are proposed key aspects of resistance. Comparative proteomic analysis of drug-resistant and -susceptible nematodes may reveal protein profiles reflecting drug-related phenotypes. Using the gastro-intestinal nematode, Haemonchus contortus as case study, we report the application of freely available expressed sequence tag (EST) datasets to support proteomic studies in unsequenced nematodes. EST datasets were translated to theoretical protein sequences to generate a searchable database. In conjunction with matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS), Peptide Mass Fingerprint (PMF) searching of databases enabled a cost-effective protein identification strategy. The effectiveness of this approach was verified in comparison with MS/MS de novo sequencing with searching of the same EST protein database and subsequent searches of the NCBInr protein database using the Basic Local Alignment Search Tool (BLAST) to provide protein annotation. Of 100 proteins from 2-DE gel spots, 62 were identified by MALDI-TOF-MS and PMF searching of the EST database. Twenty randomly selected spots were analysed by electrospray MS/MS and MASCOT Ion Searches of the same database. The resulting sequences were subjected to BLAST searches of the NCBI protein database to provide annotation of the proteins and confirm concordance in protein identity from both approaches. Further confirmation of protein identifications from the MS/MS data were obtained by de novo sequencing of peptides, followed by FASTS algorithm searches of the EST putative protein database. This study demonstrates the cost-effective use of available EST databases and inexpensive, accessible MALDI-TOF MS in conjunction with PMF for reliable protein identification in unsequenced organisms.

Hierarchical Clustering of Shotgun Proteomics Data

A new result report for Mascot search results is described. A greedy set cover algorithm is used to create a minimal set of proteins, which is then grouped into families on the basis of shared peptide matches. Protein families with multiple members are represented by dendrograms, generated by hierarchical clustering using the score of the nonshared peptide matches as a distance metric. The peptide matches to the proteins in a family can be compared side by side to assess the experimental evidence for each protein. If the evidence for a particular family member is considered inadequate, the dendrogram can be cut to reduce the number of distinct family members.

Short Regions of Sequence Identity Between the Genomes of Bacteria and Human

The interaction between bacteria and human is still incomplete. With the recent availability of many microbial genomes and human genome, as well as the series of basic local alignment search tool (BLAST) programs, a new perspective to gain insight into the interaction between the bacteria and human is possible. This study is to determine the possibility of existence of sequence identity between the genomes of bacteria and human, and try to explain this phenomenon in term of bacteriophages and other genetic mobile elements. BLAST searches of the genomes of bacteria, bacteriophages, and plasmids against human genome were performed using the resources of the National Center for Biotechnology Information (NCBI). All studied bacteria contain variable numbers of short regions of sequence identity to the genome of human, which ranged from 27 to 84 nt. They were found at multiple sites within the human genome. The short regions of sequence identity existed between the genomes of bacteria and human, and a hypothesis that viruses, especially bacteriophages, might play a significant role in shaping the genomes of bacterial and human, and contribute to the short regions of sequence identity is developed.

Finding similar nucleotide sequences using network BLAST searches.

The Basic Local Alignment Search Tool (BLAST) is a keystone of bioinformatics due to its performance and user-friendliness. Beginner and intermediate users will learn how to design and submit blastn and Megablast searches on the Web pages at the National Center for Biotechnology Information. We map nucleic acid sequences to genomes, find identical or similar mRNA, expressed sequence tag, and noncoding RNA sequences, and run Megablast searches, which are much faster than blastn. Understanding results is assisted by taxonomy reports, genomic views, and multiple alignments. We interpret expected frequency thresholds, biological significance, and statistical significance. Weak hits provide no evidence, but hints for further analyses. We find genes that may code for homologous proteins by translated BLAST. We reduce false positives by filtering out low-complexity regions. Parsed BLAST results can be integrated into analysis pipelines. Links in the output connect to Entrez, PUBMED, structural, sequence, interaction, and expression databases. This facilitates integration with a wide spectrum of biological knowledge.

Finding homologs in amino acid sequences using network BLAST searches.

AbstractThe Basic Local Alignment Search Tool (BLAST) is the most fundamental (and most misused) algorithm and software in bioinformatics/computational biology for functional assessment of unknown proteins or discovery of similar proteins with potentially common evolutionary origins. We show how to balance sensitivity with selectivity (without generating massive output) by selecting and demonstrating proper database, algorithm, and alignment display options of the user‐friendly Web sites of the National Center for Biotechnology Information (NCBI). We discuss protein query searches against protein databases and submission of all combinations of translated searches. Careful biological and statistical inferences are drawn to possible functions, taking into account the highly nonrandom nature of proteins. Guidelines for such inferences, using real‐life biological examples (e.g., protein kinases with widely distributed structural and functional domains), are provided. We show how to avoid incorrect functional inference from misleading similarities, using the divergent evolution of a serine protease domain that erodes the protease function in haptoglobins. Curr. Protoc. Bioinform. 25:3.4.1‐3.4.34. © 2009 by John Wiley & Sons, Inc.