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| # =========================================================================== | |
| # | |
| # PUBLIC DOMAIN NOTICE | |
| # National Center for Biotechnology Information | |
| # | |
| # This software/database is a "United States Government Work" under the | |
| # terms of the United States Copyright Act. It was written as part of | |
| # the author's official duties as a United States Government employee and | |
| # thus cannot be copyrighted. This software/database is freely available | |
| # to the public for use. The National Library of Medicine and the U.S. | |
| # Government have not placed any restriction on its use or reproduction. | |
| # | |
| # Although all reasonable efforts have been taken to ensure the accuracy | |
| # and reliability of the software and data, the NLM and the U.S. | |
| # Government do not and cannot warrant the performance or results that | |
| # may be obtained by using this software or data. The NLM and the U.S. | |
| # Government disclaim all warranties, express or implied, including | |
| # warranties of performance, merchantability or fitness for any particular | |
| # purpose. | |
| # | |
| # Please cite the author in any work or product based on this material. | |
| # | |
| # =========================================================================== | |
| The NCBI SRA ( Sequence Read Archive ) | |
| Contact: sra-tools@ncbi.nlm.nih.gov | |
| http://trace.ncbi.nlm.nih.gov/Traces/sra/std | |
| Stand-alone BLAST searches against SRA runs in their native format. | |
| ------------------------------------------------------------------- | |
| A stand-alone blastn application to perform BLAST searches directly against | |
| native SRA files is included in this distribution. This application has been | |
| tested in-house at the NCBI, but has not been heavily used, so this should be | |
| considered a preliminary (alpha) release to a few experienced users. A 64-bit | |
| LINUX application has been built for this testing. | |
| The application is called "blastn_vdb". | |
| The application can be invoked in much the same manner as the standard | |
| blastn application: | |
| 1) blastn_vdb -help or blastn_vdb -h will produce usage messages. | |
| 2) The BLAST+ command-line manual at http://www.ncbi.nlm.nih.gov/books/NBK1763/ | |
| provides more details on the options, though not all blastn options are | |
| available with blastn_vdb. Some options simply do not apply to sequences in SRA | |
| (e.g., -gilist is missing as these sequences have not been assigned GI's). Some | |
| options have not yet been implemented (e.g., -num_threads is currently disabled). | |
| To search cached or on-demand SRA objects. | |
| ------------------------------------------ | |
| An example blastn_vdb command-line would be: | |
| ./blastn_vdb -db "ERR039542 ERR047215 ERR039539 ERR039540" -query nt.test -out test.out | |
| The file nt.test contains the query in FASTA format, and it will be searched against | |
| the reads in runs with accessions ERR039542 ERR047215 ERR039539 ERR039540. | |
| If you have not already downloaded these objects using the vdb "prefetch" tool, | |
| they will be retrieved on-demand from NCBI under standard configuration. For | |
| alternative configuration information, please see the "README-vdb-config" file | |
| in this distribution. | |
| Searching with manually downloaded files. | |
| ----------------------------------------- | |
| If you have manually downloaded files, e.g. via aspera or wget, etc., they may | |
| be referred to as "local" files. You can pass one or more file paths to be used | |
| collectively as the database. In this case the blastn_vdb command-line would be: | |
| ./blastn_vdb -db <SRR_file> -query <input_file> -out <output_file> | |
| Where | |
| <SRR_file> is the path (relative or absolute) and name of the SRRxxxxx file | |
| <input_file> is a fasta file containing the sequence(s) to be BLASTed | |
| <output_file> is the name specified for the output report of the blast search. | |
| Example: | |
| ./blastn_vdb -db ./subdir/ERR039542.sra -query nt.test -out test.out | |
| Querying multiple SRR files simultaneously: | |
| ./blastn_vdb -db "<SRR_file1> <SRR_file2> <SRR_file3>" -query <input.fa> -out <output_file> | |
| Enclose the group of files to be included in the search set in "quotes", e.g. | |
| "./SRR_file1.sra ./SRR_file2.sra ./SRR_file3.sra" | |
| Example: | |
| ./blastn_vdb -db "./ERR039542.sra ./ERR047215.sra ./ERR039539.sra ./ERR039540.sra" -query nt.test -out test.out | |
| Caveats | |
| ------- | |
| There are some limitations on the currently available application: | |
| 1) Individual SRA data files containing more than 2 billion reads are not yet supported. For a | |
| paired-end experiment this is actually a limitation of about 1 billion "spots". | |
| 2) Compressed SRA ("cSRA") is not yet fully supported. Currently, only the | |
| unaligned fraction of reads are searched. Compressed SRA are runs containing | |
| alignments (e.g., ERR230455). Runs can be checked with "vdb-dump" to report if | |
| they contain alignment information: | |
| $ vdb-dump -E ERR230455 | |
| enumerating the tables of database 'ERR230455' | |
| tbl #1: PRIMARY_ALIGNMENT | |
| tbl #2: REFERENCE | |
| tbl #3: SEQUENCE | |
| 3) You may need to prefix "./" to the run name for files in your current | |
| directory. | |
| 4) The blast_formatter is not currently able to read native SRA files, so | |
| reformatting of results saved as a blast archive is not yet supported. | |
| Common errors and fixes. | |
| ------------------------ | |
| 1) Failure to provide relative path to manually downloaded SRR file: | |
| ./blastn_vdb -db SRR770754.sra -query srr770754_test.fa -out test.out | |
| Error: NCBI C++ Exception: | |
| "vdb2blast_util.cpp", line 253: Error: ncbi::CVDBBlastUtil::x_MakeSRASeqSrc() | |
| - VDB BlastSeqSrc construction failed: Failed to add any run to VDB runset: unsupported while allocating | |
| Fix: | |
| Include relative (e.g., "../" or "./") or absolute (e.g., "/home/user/SRA_BLAST_data/") file path with SRR file |