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NCBI GenBank has been designated as the international repository for Swine Flu (H1N1) sequence and related clinical information. In a tight cooperation between the CDC nationally and the WHO internationally, NCBI has been uploading genome and gene sequence and related information for each separately identified clinical isolate in “real-time” . To enable ready retrieval of the sequence data by those not familiar with the nuances of searching and retrieving biosequence data, NCBI has created a new set of access and retrieval tools.  The tight cooperation between the CDC and WHO to make H1N1 sequence data immediately available has been complemented by the rapid publication of  H1N1 influenza research articles by  journals such as The New England Journal of Medicine and Science.  The result has been an unprecedented publication of data, information, and tools related to manipulating the H1N1 genome and its sequences.  NCBI currently archives >19,400 H1N1 DNA sequences, almost 25,000 protein sequences of  the H1N1 genome.  Protein and domain structures are being added regularly. Truly translating between first the bed to the bench and then back again, the result has been the rapid advancement of vaccine and diagnostics R&D.  Below is a short list of highlighted swine flu bioinformatics resources, including diagnostic sequence and gene primer set  information. As we come into the flu season, the UB Health Sciences Library will update this posting as needed.

NCBI Resources

1: The WHO-designated 2009 Pandemic H1N1 diagnostic gene set.

The WHO has identified eight segments of the earliest H1N1 California isolate as diagnostic for the new H1N1 strain. They have been given NCBI GenBank accession numbers FJ966079-FJ966086.

• To download the entire set of 8 records, go to NCBI Nucleotide and copy/paste the following into the query box to run the search: FJ966079:FJ966086[Accession]
• To match the name of a diagnostic gene to its corresponding accession number, download the WHO Viral gene sequences to assist update diagnostics for influenza A (H1N1)_GenBank Accession PDF document. (Clicking on this link will launch the PDF download immediately). You can then run a search for a specific set of diagnostic gene sequences by ORing the necessary accession numbers together in the NCBI Nucleotide search box in the following format: FJ966079 OR FJ966084.

2: The NCBI H1N1 2009 Pandemic GenBank Resource web page is a component of NCBI’s much larger Influenza Viral Resource.  From the H1N1 Pandemic GenBank resource you can:

• Access the GenBank record for any of the 2009 Pandemic isolates uploaded to NCBI. For each isolate, you can open the record of any of the 8 diagnostic genes if they are available. Make sure you click on the at the bottom of the web page to fully open ALL available records, not just the most recent ones.
• BLAST your sequence against all influenza sequences, or check only the Influenza A box at the bottom of the web page to BLAST only against H1N1 sequences.
• An Advanced Query Builder has just been released that includes such features as restricting searches to a specific host, diagnostic gene set or geographical region. See the tutorial for instructions.

3. The NCBI Index of H1N1 and its various isolates

NCBI collates all information on organisms into its central Taxonomy Browser,  including for the H1N1 influenza virus. Click on the link of the desired isolate to open its specific webpage at the Index, or click on the H1N1 subtype link to view all the records of H1N1 at NCBI. In the upper right-hand corner of each webpage will be a table with links to each isolate’s nucleotide and protein records. If you choose the H1N1 subtype index, table includes links specifically to DNA, protein, structure, domain, PubMed Central publications and more. The information available here is contextual. What links exist are determined by what records are available for each isolate and will change over time.

PCR and Priming Protocols and Related Information

• CDC protocol of realtime RTPCR for influenza A (H1N1)
• WHO swine genome sequencing primer sets and protocols
• Instruction on obtaining CDC’s realtime PCR (RT_PCR) diagnostic kits for detecting Swine Flu
• Gene sequences of the reassortant candidate vaccine viruses for the novel influenza A (H1N1)
• Influenza A Virus Primer Sets from the J. Craig Venter Institute
  • This resource contains the primer sets for various Influenza A strains and their clinical isolates. Scroll to locate either the human or swine H1N1 primer sets.  They will download as an Excel file to your hard drive.

Additional H1N1 Resources:

H1N1 Structural Protein Structure Model Database at the Riken National Institute of Japan contains a summary of the predicted 3D structures of novel influenza (swine-origin influenza A/H1N1) proteins. These proteins are necessary for virus proliferation, and make ideal targets for the development of anti-influenza drugs. It also contains a summary of the amino acid differences between the swine flu proteins and those in the Protein Databank which are generated by RPS-BLAST. The origin of the sequence data for modeling is from NCBI which is updated daily by Riken if needed. You can view and compare all these data from each novel H1N1 gene of any clinical isolate simultaneously on the same web page.

NIAID Influenza Research Database (IRD) is an integrated resource for the analysis and visualization of influenza viral proteins, genes and strains. The resource contains several types of viral search and analysis tools including mutation analysis, phenotype, protein structure and surveillance data searches. IRD has built-in H1N1 to its search and visualization resources. Just make sure to specify the H1N1 viral subtype if the option is available. A tutorial is also available. (Clicking on this link will launch the PDF download immediately).

Read over the workshop descriptions below and decide which  work for you . You can then register for the workshops here, or, more importantly contact me at drein@buffalo.edu to arrange for the workshop to come on-site, tailored to your specific needs for your research laboratory, seminar, course, or group of any kind.

Don’t want BLAST? Want EndNote or something else instead? The BLAST Short Course is part of the HSL workshops offered every semester. If you are interested in EndNote, tenure metrics and more, take the opportunity to look over our other offerings and register for these courses as well. You can read about all of the HSL’s workshops, and then register for them, on HSL’s workshop webpage.

WORKSHOP 1: BLAST

BLAST, the Basic Local Alignment Search Tool from the National Center for Biotechnology Information (NCBI) at NIH, has become the major bioinformatics search tool to locate similar sequences to DNA or protein sequences. Proper analysis of BLAST results simultaneously provides insights into gene/protein structure and functions for unknown sequences while aiding in defining future experiments.  A combination handout/workbook will be given to workshop participants that will cover the workshop material.

Following this workshop you will be able to:

• Search with BLAST.
• Decipher and analyze search results, including E values.
• Understand BLAST algorithmic interface options.
• Know the strengths and limitations of BLAST.
• Know how to choose between:
  • Basic BLAST options (blastp, blastn, tblastn, blastx, etc.).
  • Genomic BLAST.
  • Specialized BLAST options (microarray, immunoglobulin, high-throughput sequencing, etc.).
• Use BLAST to align and compare sequences of your choice.
• Run mutation BLAST with either protein or nucleotide sequences.
• Edit, limit, and re-format both BLAST searches and results.
• Link-out from BLAST results to other related NCBI information for a given sequence.
• Manage BLAST search results.

TIMES AND LOCATIONS:

September 22, 2009:  NORTH Campus— Room 212, Teaching & Learning Center, Capen Hall.
September 21, 2009:  SOUTH Campus—Media Instruction Room, Health Sciences Library.

REGISTER

WORKSHOP 2: BLAST TIPS AND TRICKS

This two-hour practical hands-on workshop will focus to adjusting those BLAST algorithmic parameters which act as filters for BLAST, along with other “advanced commands” that can be used to enhance BLAST results. The workshop will concentrate primarily to nucleotide and protein BLASTing, but will cover other specialized BLAST searches based upon participant interest and available time. The new NCBI multiple alignment tool, COBALT, will also be introduced. A combination handout/workbook will be given to workshop participants that will cover the workshop material.

Following this workshop you will be able to:

• Constrain BLAST searches to specific sequence segments of your query using a variety of different methods.
• Limit the initial BLAST search to specific organisms, tissues of expression, molecule types or databases.
• Restrict the BLAST results to specific organisms, tissues of expression, molecule types or databases to make your analysis easier.
• Use filtering options to lower the number of statistically significant but meaningless hits to query sequences.
• Batch BLAST.

TIMES AND LOCATIONS:

September 28, 2009:  SOUTH Campus—Media Instruction Room, Health Sciences Library.
September 29, 2009:  NORTH Campus— Room 212, Teaching & Learning Center, Capen Hall.

REGISTER

WORKSHOP 3: BEYOND BLAST: POWERING UP YOUR SEQUENCE SIMILARITY SEARCHES WITH NCBI PSI-BLAST AND PHI-BLAST

Position-Specific Iterative (PSI) BLAST is a protein sequence search method that generates a protein search profile in place of the original BLAST algorithm. More importantly, you the searcher, choose the sequences you need to build the search profile for subsequent iterations. Pattern Hit Initiated (PHI)-BLAST BLAST is similar, with the notable exception that you can specify a protein motif or repeating protein element to build your protein profile. Both PSI-BLAST and PHI-BLAST provide a means to detecting distant functional relationships between proteins that are beyond the capability of BLAST. They are also the method of choice if “traditional” BLAST is returning too few of hits or unannotated hits. The recently re-formatted NCBI PHI- and PSI-BLAST interface and algorithms now put these powerful tools into the hands of all BLAST users. Additionally, both PSI- and PHI-BLAST are integrated with any 3D structures returned within the hits, aligning sequences recovered from a search to the target sequence.  This two-hour workshop will work through the underlying concept of scoring matrices upon which PHI- and PSI BLAST are built upon and provide practical hands-on experience in PHI- and PSI-BLASTing. A combination handout/workbook will be given to workshop participants that will cover the workshop material.

Following this workshop you will be:

• Understand scoring matrices and their use in BLAST, PSI-BLAST and PHI-BLAST
• Know when and how to run PHI-BLAST and PSI-BLAST
• Able to link-out from PHI-BLAST results to discover gene and protein structural information for your query.

October 5, 2009:  NORTH Campus— Room 212, Teaching & Learning Center, Capen Hall.
October 6, 2009:  SOUTH Campus—Media Instruction Room, Health Sciences Library.

REGISTER

The New NIH RePORTER Grant Search Engine

The original functionality of CRISP will be retained in RePORTER but significantly enhanced. This change was mandated as part of the legislative NIH Reform Act of 2006, which requires NIH to provide access to publications and patents resulting from NIH expenditures, including grant awards. When RePORTER goes completely live on September 9, new features will include:

• additional query fields
• ability to sort hit lists in a variety of ways
• download results to Excel
• any publications and/or patents resulting from grants and projects will be linked directly from within the hits list from several sources which currently include:
  • PubMed
  • PubMed Central
  • US Patent & Trademark Office Patent Full Text and Image Database
  • extramural funding available from the NIH eRA databases
  • intramural funding from the NIH Intramural Database

In turn, RePORTER itself will be embedded within the much larger RePORT web resource that provides additional search engines for:

• NIH Reports
  • Using pull-down menus, you can search for data from within NIH Reports. For example, you can craft a search to return data on success rates for R01 applications submitted to all NIH Institutes and Centers in fiscal year 2008.
• Categorical Spending
  • Currently, you can search >200 diseases or conditions, generating a list funded grant information including project title, amount awarded, PI and more
• NIH Data Book
  • Provides basic summary statistics on extramural grants, contract awards, grant applications, scientific workforce, etc. and more

The new RePORT web site, as well as the RePORTER search engine, will undergo a series of iterations throughout fiscal year 2010 as NIH continues to mature the resource to include new indexing schemes, more extensible search options and new features related to specific diseases and other conditions.

Additional related links:

• RePORT Overview
• RePORT Tutorial: A Flash-based tutorial that reviews basic features of the RePORT web site. The tutorial is divided into separate learning modules. You can choose which module you wish to view in whatever order you wish or let the tutorial run its pre-selected course set by NIH.
• RePORT FAQS

As its name implies, Genome Workbench is an integrated, customizable application for viewing and analyzing sequence data. One of its strengths is its ability to import publically available sequence data and mash it up with your own imported private data. There is no online version of Genome Workbench. You must download and install the program onto your computer, which also means your private data stays private to your computing environment. Once you have downloaded it, run the Tutorial 1: Basic Operation, an excellent set of exercises which will acquaint you with the interface and options of the Workbench. You can follow the tutorials searches and data uploads, or substitute your own. A strong point of the Workbench is its full set of tutorials and practice datasets ranging from basic to advanced and include working with variation and alignments.

Some Genome Workbench Features:

• Organizes data into Projects of your choosing (specific genes/sequences and their transcripts, regulatory elements, proteins, SNPs, STSs, etc.).
  • Projects are then collected into Workspaces (genes from different organisms, sequences correlated to a specific disease, your data chunks from the same organism, etc.)
• Zoomable graphics mode.
  • Explore sequence data at different levels of detail, ranging from individual exons to genomic contexts, marching up and down chromosomes as needed.
• View data as alignments, phylogenetic trees, graphically or as tables.
• BLAST from within graphics mode.
• Multiple options to align sequences including  ClustalW, MUSCLE.
• Analyze variation, including SNPs.
• Import Genome Wide Association Studies (GWAS) data.
• Link-out service from within Workbench, including linking from sequences within Workbench to:
  • NCBI Gene Database
  • Contig Overlap to evaluate abutting genomic sequences.
• Click here to begin an immediate download of a NCBI PowerPoint presentation on the Genome Workbench which provides many more screenshots and a full overview.

Additional related links:

• Genome Workbench FAQ, including the four mailing list servs that support it.
• Help home page.