Program Option for Netblast (blastcl3)
Tao Tao, Ph.D. User Service NCBI, NLM, NIH
TOC

- 1. Introduction
- 2. Installation and setup
- 3. Firewall settings
- 4. Options and their accepted values
- 5. Practical usage examples
  - 5.1 General nucleotide searches
  - 5.2 General protein searches
  - 5.3 Translated BLAST searches
    - 5.3.1 blastx
    - 5.3.2 tblastn
    - 5.3.3 tblastx
  - 5.4 Genome BLAST searches
    - 5.4.1 Microbial Genomes and Other Eukaryote Genomes
    - 5.4.2 Higher Genomes
- 6. Trouble shooting and technical assistance
  - 6.1 Errors and warnings
  - 6.2 Technical assistance

1. Introduction

NCBI BLAST web server provides a convenient and user friendly way for individuals to search their queries against different public sequence databases. This server, however, does have some limitation. For example, one will not be able to perform large scale batch searches from most of the BLAST pages and the program selection for some of the available databases is limited. BLAST client provides a way to circumvent those limitations.

The client bypasses web browser and interacts directly with the NCBI BLAST server that powers the NCBI web BLAST service (www.ncbi.nlm.nih.gov/BLAST/). It performs the batch search with multiple sequences by taking one query sequence from the input file (with multiple FASTA formatted sequences), formulating the search according to the command line, and sending the search through the internet connection to NCBI BLAST server for processing. The program receives the search result from blast server and saves it to a local file specified by the command line. The program loops through all the queries in the input file till all are searched.

This program has no graphic user interface (GUI) and must be executed from command line under a terminal window. Users control the program through command line options. Detailed list of command line options are in Section 4. For usages and situation examples, see Section 5.

2. Installation and setup

NCBI provides BLAST client as an archive separate from that of the standalone (blast initialed) or server blast (wwwblast initialed) package. This archive is available for common platforms as netblast initialed files. They can be found at:

> ftp.ncbi.nlm.nih.gov/blast/executables/LATEST/

For Linux or Unix environment, installation is straight forward. One can place the archive in a desired directory and extract the archive using the following command line:

> tar zxvf netblast-##-**.tar.gz

The resulted netblast-## directory contains bin, doc, and data subdirectories. The program, blastcl3, is under the bin subdirectory. The matrices BLAST needs for protein alignments are under the data subdirectory, while the doc subdirectory contains netblast.html (this file) and firewall.html with more information on configuration under firewall settings.

The package for Windows can be extracted using WinZip. It does not have this directory structure.

3. Firewall settings

The setup for NCBI network clients has been greatly simplified. If you are not behind a firewall no further action is required. If you are behind a firewall, and already use Sequin or Entrez, or if your system administrator has already performed the setup, then you should be able to start performing searches immediately after installation. Otherwise, your will need to make sure that the following IP address/port combinations are open in the firewall configuration.

Table 3. Firewall Ports Needed by BLAST Client for NCBI Connection
IP Address | Port Number
130.14.29.112 | 5861
130.14.29.112 | 5862
130.14.29.112 | 5863
Note Please refer to 'firewall.html' included in the package for details.

In addition to this, you also need to create an .ncbirc file placed in the home directory to instruct blastcl3 how to make the connection to NCBI. For PC running Windows, the file is named ncbi.ini which should be placed under the windows directory. A sample .ncbirc file is provided in the text box below for your reference.

> [NCBI]
> DATA=/home/johndoe/netblast-2.2.12/data
> [CONN]
> FIREWALL=TRUE
> [NET_SERV]
> SRV_CONN_MODE=SERVICE

As an alternative to blastcl3, NCBI BLAST web server also supports URL API, which uses URL encoded command to interact with Blast.cgi directly to "Put" search requests or to "Get" search results. For details on the standard commands, please refer to the online document at:

> www.ncbi.nlm.nih.gov/blast/Doc/urlapi.html

4. Options and their accepted values

As mentioned before in Section 1, blastcl3 has no GUI and works only under a command terminal. Users execute the the program by issuing command lines, and controls the way blast search is done through options in the command line. The command line options for this program are listed here individually below. The options commonly adjusted during actual searches are: -i, - d, -p, -o, -e, -F, -u, -b, -v, -m, and -n. The first four are mandatory.

Table 4.1
Option | -p
Function | Specifies which program to run
Default | None, mandatory
Input Format | String
Example | To run blastn program use: -p blastn
Note | Program string options and type of search they specify
Program | Query | DB
blastn | nucleotide | nucleotide
blastp | protein | protein
blastx | nucleotide, translated | Protein
tblastn | protein | nucleotide, translated
tblastx | nucleotide, translated | nucleotide, translated

Table 4.2
Option | -d
Function | Specifies database(s) to be searched
Default | nr
Input Format | String
Example | One can search against multiple databases can be specified in command line. To search nr and est at together use: -d "nr est"
Note | Be conservative. Search against large databases may not complete due to CPU time limit, which is set at one hour.

Table 4.3
Option | -i
Function | Specifies input query file
Default | stdin
Input Format | String, mandatory
Example | To use sequences from query.txt as query, use -i query.txt
Note | One should use the complete file name WITH its extension. To use stdin default, omit the -i and redirect using: < mito.txt

Table 4.4
Option | -e
Function | Specifies Expect value cutoff
Default | 10
Input Format | Real
Example | To make the search more stringent, one can use: -e 0.001
Note | Accepted formats are integer, fraction, decimal, exponential and scientific notation. To set the cutoff to 2×10-20, use -e 2e-20

Table 4.5
Option | -m
Function | Specifies alignment view option
Default | 0
Input Format | Integer
Example | To display the result in XML form use: -m 7
Note | Option values and the output formats they specify
0 | Pairwise
1 | query-anchored showing identities
2 | query-anchored no identities
3 | flat query-anchored, show identities
4 | flat query-anchored, no identities
5 | query-anchored no identities and blunt ends
6 | flat query-anchored, no identities and blunt ends
7 | XML Blast output
8 | tabular (not post processing)
9 | tabular with comment lines (post-processed, sorted)
10 | ASN, text
11 | ASN, binary

Table 4.6
Option | -o
Function | Specifies result output file
Default | stdout (print to screen)
Input Format | String [file name]
Example | To save result in out.txt use: -o out.txt
Note | -p, -i, -d, -o are the core options needed for a blastcl3 search.

Table 4.7
Option | -F
Function | Specifies which filter(s) to use to mask query sequence
Default | T (DUST for nucleotide, SEG for protein)
Input Format | String
Example | To filter low complexity and lookup table only, use: -F "m L"

Note

Accepted strings: T, F, D, L, R, V, S, C, and m.
m in -F stands for masking for lookup table only, which enables blast to
display the masked region in the alignment. L stands for Low complexity,
D stands for DUST. R stands for human Repeats, V stands for Vector.
S stands for SEG, which has other user specifiable values:
-F "S 10 1.0 1.5" SEG filter: window=10; low cut=1; high cut=1.5.
C stands for COIL, which also has user specifiable values:
-F "C 28 40 32" COIL filter: window=22; cutoff=40; linker=32.
To run SEG and COIL filter together, use: -F "S; C"
To mask lookup table only, add m: -F m "S; C"
To mask repeat sequences use: -F R or -F "m R"
To combine all together, use: -F "m L;R"
To mask vector filter, use: -F V
To call rodent repeat filter, use: -F "R -d rodent.lib"

Table 4.8
Option | -G
Function | Cost to open a gap
Default | 0
Input Format | [Integer]
Example | To increase the gap open penalty to 10, use: -G 10
Note | Zero invokes default (5) for blastn. It varies for blastp, blastx, tblastn, and tblastx. In protein searches, only a controlled set of -G/-E value pairs are acceptable for a given scoring matrix.

Table 4.9
Option | -E
Function | Cost to extend a gap
Default | 0
Input Format | [Integer]
Example | To increase the gap extension penalty to 4, use: -E 4
Note | Zero invokes default or 2 for blastn. Varies for blastp, blastx, tblastn, and tblastx. In protein searches, only a controlled set of -G/-E value pairs are acceptable for a given scoring matrix.

Table 4.10
Option | -X
Function | X dropoff value for gapped alignment (in bits)
Default | 0
Input Format | [Integer]
Example | To increase the gapped alignment dropoff to 40, use: -X 40
Note | Gapped Alignment Dropoff Default Setting (in bits)
Program | blastn | megablast | tblastx | others
Value | 30 | 20 | 0 | 15

Table 4.11
Option | -I (capital i)
Function | Show GI in definition line
Default | F
Input Format | [T/F]
Example | To activate the GI display use: -I T
Note | Sample display: T: gi|223046|prf||0410468A... F: prf||0410468A...

Table 4.12
Option | -q
Function | Penalty for a nucleotide mismatch
Default | -3
Input Format | [Integer]
Example | To set penalty to -2, use: -q -2
Note | For blastn only, different -r/-q ratios are optimal for aligning sequences with different percentage of similarities.

Table 4.13
Option | -r
Function | Reward for a nucleotide match
Default | 1
Input Format | [Integer]
Example | To increase the reward to 2, use: -r 2
Note | For blastn only. Others use external scoring matrix to determine this. See -M table in blastall for more details.

Table 4.14
Option | -v
Function | Number of database sequences to show one-line descriptions for
Default | 500
Input Format | [Integer]
Example | To increase the descriptions displayed to 1000 use: -v 1000
Note | Web counterpart is "Descriptions"

Table 4.15
Option | -b
Function | Number of sequences with alignments to show
Default | [Integer]
Input Format | 250
Example | To increase the alignment displayed to 1000 use: -b 1000
Note | Upper limit is 200000. Web counterpart: "Alignments". This is NOT the total number of alignment segments or high scoring pairs (HSPs). Rather it is the number of database sequences with HSP(s) to the query.

Table 4.16
Option | -f
Function | Threshold for extending hits
Default | 0
Input Format | Integer
Example | To increase this threshold to 15, use: -f 15
Note | Default if set to zero, not used by blastn or megablast. Extension Threshold Default Settings
Program | blastp | blasn | blastx | tblastn | tblastx | megablast
Value | 11 | 0 | 12 | 13 | 13 | 0

Table 4.17
Option | -g
Function | Perform gapped alignment
Default | T
Input Format | [T/F]
Example | To do only ungapped alignment, use: -g F
Note | Default is gapped alignment, not available with tblastx.

Table 4.18
Option | -Q
Function | Query genetic code to use
Default | 1
Input Format | [Integer]
Example | To set the genetic code (translation table) to 14, use: -Q 14
Note | This determines which translation table to use on query in translated blastx and tblastx searches. Default is universal codon.

Table 4.19
Option | -D
Function | DB Genetic code
Default | 1
Input Format | [Integer]
Example | To set the genetic code (translation table) to 14, use: -D 14
Note | Determines which translation table to use for the database in tblastn and tblastx search. See details at: www.ncbi.nlm.nih.gov/Taxonomy/Utils/wprintgc.cgi?mode=c

Table 4.20
Option | -a
Function | Number of processors to use
Default | 1
Input Format | [Integer]
Example | To change this to two CPUs, use: -a 2
Note | From 1 up to the number of CPUs available.

Table 4.21
Option | -O
Function | To save SeqAlign object
Default | N/A
Input Format | String [File Out]
Example | To save SeqAlign object to blast_seqalign, use: -O blast_seqalign
Note | User can use the output to reformat the result into different format using NCBI toolkit function. See /blast/demo/ subdirectory for more information.

Table 4.22
Option | -J
Function | Believe the query definition line
Default | F
Input Format | [T/F]
Example | To set this to true, use: -J T
Note | Default set to false since query file definition lines may not follow NCBI convention.

Table 4.23
Option | -M
Function | Protein scoring matrix to use
Default | BLOSUM62
Input Format | [String]
Example | To change this to PAM30, use: -M PAM30
Note | Accepted value: BLOSUM45, BLOSUM62, BLOSUM80, PAM30, or PAM70.

Table 4.24
Option | -W
Function | Word size
Default | 0
Input Format | [Integer]
Example | To set word size to 32, use: -W 32
Note | Word size setting for different programs
Program | blastn | megablast | all others
Value | 11 | 28 | 3

Table 4.25
Option | -z
Function | Effective length of the database
Default | 0
Input Format | [Real]
Example | To set this to 10000000, use: -z 10000000
Note | Use zero for the actual database size.

Table 4.26
Option | -K
Function | Number of best hits from a region to keep
Default | 0
Input Format | [Integer]
Example | To keep 200 hits, use: -K 200
Note | This selects the specified number of best hits for a given region of the query for further evaluation. Off by default, 100 recommended if used.

Table 4.27
Option | -P
Function | Use multiple hit
Default | 0
Input Format | Integer
Example | To do single hit, use: -P 1
Note | Zero is for multiple hit, 1 for single hit. Not applicable to blastn.

Table 4.28
Option | -Y
Function | Effective length of the search space
Default | 0
Input Format | [Real]
Example | To set this to 10000000, use: -Y 10000000
Note | This is the product of effective query length and effective database length - actual length corrected for edge effects. Use zero for actual size.

Table 4.29
Option | -S
Function | Strands of the nucleotide query to use in the search
Default | 3
Input Format | [Integer]
Example | To search with the reverse complement strand only, use: -S 2
Note | -S Input Code And Meaning for blastn, blastx, and tblastx.
Meaning | Input | Reverse complement | Both
Value | 1 | 2 | 3

Table 4.30
Option | -T
Function | Produce HTML output
Default | F
Input Format | [T/F]
Example | To generate HTML formatted output, use: -T T
Note | With -T T, if the database is from NCBI, BLAST will hot link matched subject sequences to their actual entries in Entrez.

Table 4.31
Option | -u
Function | Restrict search of database to the subset satisfying the query
Default | N/A
Input Format | [Entrez Term] in quotes
Example | To restrict entries to mRNA use: -u "biomol_mrna[prop]"
Note | Argument is a set of Entrez query terms. BLAST server will use the terms to retrieve a list of GI numbers and restrict the BLAST search to entries specified by the list. Make sure valid terms are used. For example, it does not make sense to restrict a search to genomic sequences while searching against the est database. For details, see Entrez Help

Table 4.32
Option | -U
Function | Use lower case filtering of FASTA sequence
Default | F
Input Format | [T/F]
Example | To turn lowercase filter on, use: -U T
Note | Make sure that the query sequences are in UPPERCASE and only the filtered portions are in lowercase.

Table 4.33
Option | -y
Function | X dropoff value for ungapped extensions (in bits)
Default | 0
Input Format | [Real]
Example | To increase the dropoff to 25, use: -y 25
Note | Default setting for ungapped alignment X dropoff (-y, in bits)
Program | blastn | megablast | others
Value | 20 | 10 | 7

Table 4.34
Option | -Z
Function | X dropoff value for final gapped alignment (in bits)
Default | 0
Input Format | [Integer]
Example | To increase this dropoff to 60, use: -Z 60
Note | Large dropoff value settings may help generate longer alignment. Default setting for ungapped alignment X dropoff (-Z, in bits)
Program | blastn | megablast | tblastx | all others
Value | 50 | 50 | 25 | 0

Table 4.35
Option | -R
Function | Run rpsblast search
Default | F
Input Format | [T/F]
Example | To run rpsblast search, use: -R T
Note | Performs rpsblast search against CDD database. Requires an appropriate -d input. See "Remote Accessible BLAST Databases" for more information.

Table 4.36
Option | -n
Function | Enable megablast search
Default | F
Input Format | [T/F]
Example | To enable megablast search, use -n T
Note | Invokes megablast algorithm when set to T. -W will default to 28 and queries will be concatenated. This will help speed up the search at the expense of search sensitivities.

Table 4.37
Option | -L
Function | Location on query sequence
Default | N/A
Input Format | [String]
Example | To search with 100 to 400 of a query, use: -L "100,400"
Note | In -L "100,400", 100 is the start and 400 the end.

Table 4.38
Option | -A
Function | Multiple hits window size
Default | 0
Input Format | [Integer]
Example | To increase the window size to 50, use: -A 50
Note | Default -A setting for different programs
Program | blastn | megablast | all others
Value | 0 | 0 | 40

Table 4.39
Option | -w
Function | Frame shift penalty
Default | 0 (no penalty)
Input Format | [Integer]
Example | To set OOF penalty to 10, use: -w 10
Note | Non-zero invokes OOF (Out Of Frame) algorithm for blastx.

Table 4.40
Option | -t
Function | Length of the largest intron allowed in tblastn for linking HSPs
Default | 0
Input Format | [Integer]
Example | To allow linking of HSPs 10000 letter apart, use: -t 10000
Note | Zero disables linking. Otherwise, the value specified will be used.

5. Practical usage examples

Before we get into the actual use, we need to discuss the format of the input query. The only query format blastcl3 recognizes is FASTA. In this format, the query begins with a "greater than" sign (>) initialed definition line, or defline as it is commonly known. This defline contains a basic description of the sequence, such as its source, the gene it represents, or ways to identify the sequence. It is terminated by a hard return. Actual sequence immediately follows the defline in one or more lines each terminated by a hard return. Multiple query sequences should be concatenated one after another. Sample query sequences are presented below for your reference.

    >gi|4557757|ref|NP_000240.1| MutL protein homolog 1
    MSFVAGVIRRLDETVVNRIAAGEVIQRPANAIKEMIENCLDAKSTSIQVIVKEGGLKLIQIQDNGTGIRK
    EDLDIVCERFTTSKLQSFEDLASISTYGFRGEALASISHVAHVTITTKTADGKCAYRASYSDGKLKAPPK
    PCAGNQGTQITVEDLFYNIATRRKALKNPSEEYGKILEVVGRYSVHNAGISFSVKKQGETVADVRTLPNA
    STVDNIRSIFGNAVSRELIEIGCEDKTLAFKMNGYISNANYSVKKCIFLLFINHRLVESTSLRKAIETVY
    AAYLPKNTHPFLYLSLEISPQNVDVNVHPTKHEVHFLHEESILERVQQHIESKLLGSNSSRMYFTQTLLP
    GLAGPSGEMVKSTTSLTSSSTSGSSDKVYAHQMVRTDSREQKLDAFLQPLSKPLSSQPQAIVTEDKTDIS
    SGRARQQDEEMLELPAPAEVAAKNQSLEGDTTKGTSEMSEKRGPTSSNPRKRHREDSDVEMVEDDSRKEM
    TAACTPRRRIINLTSVLSLQEEINEQGHEVLREMLHNHSFVGCVNPQWALAQHQTKLYLLNTTKLSEELF
    YQILIYDFANFGVLRLSEPAPLFDLAMLALDSPESGWTEEDGPKEGLAEYIVEFLKKKAEMLADYFSLEI
    DEEGNLIGLPLLIDNYVPPLEGLPIFILRLATEVNWDEEKECFESLSKECAMFYSIRKQYISEESTLSGQ
    QSEVPGSIPNSWKWTVEHIVYKALRSHILPPKHFTEDGNILQLANLPDLYKVFERC
    >gi|68348711|ref|NP_001234.2| tumor necrosis factor receptor 8
    MRVLLAALGLLFLGALRAFPQDRPFEDTCHGNPSHYYDKAVRRCCYRCPMGLFPTQQCPQRPTDCRKQCE
    PDYYLDEADRCTACVTCSRDDLVEKTPCAWNSSRVCECRPGMFCSTSAVNSCARCFFHSVCPAGMIVKFP
    GTAQKNTVCEPASPGVSPACASPENCKEPSSGTIPQAKPTPVSPATSSASTMPVRGGTRLAQEAASKLTR
    APDSPSSVGRPSSDPGLSPTQPCPEGSGDCRKQCEPDYYLDEAGRCTACVSCSRDDLVEKTPCAWNSSRT
    CECRPGMICATSATNSRARCVPYPICAAETVTKPQDMAEKDTTFEAPPLGTQPDCNPTPENGEAPASTSP
    TQSLLVDSQASKTLPIPTSAPVALSSTGKPVLDAGPVLFWVILVLVVVVGSSAFLLCHRRACRKRIRQKL
    HLCYPVQTSQPKLELVDSRPRRSSTQLRSGASVTEPVAEERGLMSQPLMETCHSVGAAYLESLPLQDASP
    AGGPSSPRDLPEPRVSTEHTNNKIEKIYIMKADTVIVGTVKAELPEGRGLAGPAEPELEEELEADHTPHY
    PEQETEPPLGSCSDVMLSVEEEGKEDPLPTAASGK

Note that the file containing the query sequences has to be saved as a plain text file.

The program runs under a command or terminal window. On PC the command window can be launched using "Start ► Program ► Accessories ► Command Prompt". On Mac, the Terminal program icon usually is under the Utilities folder. Double click the grey icon will launch it.

In the terminal window, cd to the directory containing the blastcl3, then run the program from there. Type "blastcl3 -" without quotes and hit return should display the command line options on the screen. On Mac and Unix/Linux platform, type "./blastcl3 -" without quotes.

Since the list of available database has increased significantly, they are removed from this file. We will document this in a separate file at a later time.

5.1 General nucleotide searches

The primary use of nucleotide BLAST search is to identify the input query by finding if exact match(es) are present in the database. This type of search also is used to identify the genomic counterpart of an input mRNA sequence or vice versa. Sometimes it is also used to search with primer pairs to identify the annealing target and possible secondary annealing sites of the primers.

For sequences from well studied model organisms, a good approach is to search against the refseq_rna or refseq_genomic database with Entrez limit. Alternatively, search against nr with or without limit to the target organism can also offer good lead.

The following example command lines search the input query file new_seq.txt against either the refseq_rna or nr database and save the result in n_refm.out and n_nr.out, respectively.

> blastcl3 -p blastn -i n_seq -p blastn -d refseq_rna -o n_refm.out
> blastcl3 -p blastn -i n_seq -p blastn -d nr -o n_nr.out

We can further restrict the search to the mouse entries in those two databases by using entrez limit and to speed up the search by invoking the megablast algorithm. The following two options will accomplish that:

> -u "mouse[organism]" -n T

For easy parsing of the search result, we can request that the result be returned in either XML or "Hit Table" (tabular) format using "-m 7" or "-m 9" without quotes in the command line.

Seaching a genomic DNA against nucleotide database, we should invoke the repeat filter to mask the repeat region and prevent BLAST program from being inundated by spurious hits to those regions. For human, this can be invoked by adding the following filter option to the command line:

> -F "m L; R"

Rodent specific repeat filter requires different filter call:

> -F "R -d rodent.lib"

Combining these together, the following command line searches the n_seq input nucleotide query file against the human subset in the refseq_genomic database with low complexity and human repeat filter and megablast algorithm. The expect value cutoff is set to 2x10-10 and the output is saved in refg.output:

> blastcl3 -i n_seq -p blastn -d refseq_genomic -u "human[orgn]" -n T -F "m L; R" -e 2e-10 -o refg.output

5.2 General protein searches

A protein BLAST search can be used to identify the input query protein or its function through matching to other known proteins and their annotation. One such database is refseq_protein. The following command line searches protein sequences in my_query against this database using blastp. The result is saved in my_output.

> blatcl3 -p blastp -i my_query -d refseq_protein -o my_output

For functional analysis, direct search against cdd database is more informative. Matches from cdd search will identify the conserved functional domain(s) present in the query. Defline and annotation from these matched domains will provide a better revelation on the function of the query. The following command line does such a search against the cdd database (-d cdd) using rpsblast (-R T):

> blatcl3 -p blastp -R T -i my_query -d cdd -o my_output

Specific search against pdb database can be used to identify existing structures with matching sequences useful for structure modeling purposes. We do not support PSI-BLAST or PHI-BLAST searches through blastcl3.

5.3 Translated BLAST searches
Translated searches can be very informative in revealing the possible function of the query since the search and alignment is performed at the protein level, which is more sensitive and biologically relevant.
5.3.1. blastx

This program searches a nucleotide query against a protein database. It first translates the query in all six frames and then searches those protein translations against the specified protein database. It is useful in identify the potential protein product(s) the query may encode and may even be able to provide information on the functions of the protein(s) should a good match to a well characterized protein can be found.

In the example command line below, we are searching the nucleotide sequences in my_query against refseq_protein. The results are saved in my_oputput file.

> blatcl3 -p blastx -i my_query -d refseq_protein -o my_output

5.3.2 tblastn

This program function searches an input query protein sequence against a target nucleotide database to find other potential protein sequences that might be encoded by those nucleotide sequences. It is a good way to find out yet unidentified homolog/paralog of a give protein query. During the search, the nucleotide database entries are first translated in all six frames. The query protein is then compared against those potential products to identify the matches.

Example given below searches the input protein query file my_query against est_human database to try to identify human est entries that may encode proteins similar to the query. The result is saved to my_output:

> blastcl3 -p tblastn -i my_query -d est_human -o my_output

5.3.3. tblastx

This program function compares all six-frame translations of an input query nucleotide against those from a nucleotide database. Since this search is very computationally expensive, we strongly recommend that you use it with caution, employ an higher search stringency, and limit the search to a smaller more specific subset of the database using entrez limit.

The following command line searches the my_query against the human genomic entries in nt database. The result is saved in my_output.

> blastcl3 -p tblastx -i my_query -d nt -u "human[orgn] AND biomol_genomic[prop]" -o my_output

Due to the heavy computation intensity, we also recommend that users set up local standalone blast to performing such searches if the search volume is large and/or the need is regular.

5.4 Genome BLAST searches

Genome BLAST pages collect the genomic sequences and other sequences specific to an organism in one place for easy access. In addition, the matches from searching these databases often contain links to the graphic display on the Genome Mapviewer for that organims. Those organism specific genomic and other sequence databases are also available for search using blastcl3 with one major difference - there will be no link to the Map Viewer.

5.4.1 Microbial Genomes and Other Eukaryote Genomes

Depending on the status of the genome, they can be finished with accompying protein data, wgs with accompanying protein data, or wgs without accompanying protein data. The database naming convention is "Microbial/Taxid". The example command line below searches the protein database for E.coli K-12 strain:

> blastcl3 -p blastp -i my_query -d Microbial/83333 -o my_output

NCBI is terminating the support for BLAST searching unfinished microbial genomic sequences through the microbial genome blast page. The recommended way is to blast against the wgs database since most of their genomic sequences are submitted to NCBI as wgs entries. The following command line example searches the wgs entries of Bacillus anthracis:

> blastcl3 -p blastn -d wgs -i my_query -o my_output -u "bacillus anthracis[orgn] AND wgs[prop]"

5.4.2 Higher Genomes

Higher genomes related databases are grouped according to orgamisms, each group has it own unique database prefix. The genome assemblies are build-specific and they are updated when now assemblies are made available. For example, the human genome database and other human specific databases have the "hs_genome/" prefix. The following example command line searches against all the available human genome assemblies:

> blastcl3 -p blastp -i my_query -d hs_genome/all_contig -o my_output

The default filter in the human genome blast page is "low complexity, human repeat, and masking lookup table only". To emmulate this, we can add -F "mL;R" into the command line.

6. Trouble shooting and technical assistance
6.1 Errors and warnings

Problems encountered while using blastcl3 can be caused by firewall configuration, internet connection interruption, or NCBI server glitches, with the firewall configuration as the most common cause. A representative error message may contain "[CONN_Open] Cannot open connection", "<<< Re-establishing NETBLAST Service >>>", or something in that order.

Adding the following two lines in the .ncbirc file will increase the timeout setting and generate more informative messages that are useful in debugging the problem:

> TIMEOUT=300
> DEBUG_PRINTOUT=DATA

Search related errors from NCBI BLAST server typically are accompanied by RID for that search. Those RIDs should be kept and sent to NCBI blast-help for trouble-shooting.

6.2 Technical assistance

If you encounter netblast problems, please report them to blast-help alias below. We recommend that you copy the error/warning messages displayed on the screen and provide detailed command line, and other relevant information. Questions or comments on this document and on BLAST in general should also be sent to blast-help alias.

> blast-help@ncbi.nlm.nih.gov

Questions on other NCBI resources should be sent to:

> info@ncbi.nlm.nih.gov