Seedtop Document

Program Options for seedtop

Tao Tao, Ph.D.
User Service
NCBI, NLM, NIH

Table of Content

1. Introduction
2. Setup
3. Program options
4. Practical Usage
5. Technical Support

seedtop is a little-known program found in the NCBI standalone blast package, whose main function is to search for patterns in an input sequence or database. It has four modes of usage, which are referred to as "subprograms": two for pattern searches from a input query or database only and two for pattern initiated sequence alignment. The following table lists these subprograms, their functions, and required inputs.

Table 1.1 Subprogram Fucntion of seedtop
Program Call ¹	Functions	Required Inputs
-p patmatch	Search for patterns in an input sequence	Pattern (-k) and sequence (-i)
-p pattern	Search for patterns in an input database	Same as above
-p patseed	Search for patterns in the query and align the query against a database	Pattern (-k), input sequence (-i), and target database (-d)
-p seed	Search for specific pattern in the query and align the query against a database	Same as above ²
NOTE: ¹ The program strings listed are for nucleotide searches. For protein searches, add lowercase p to the program name. ² The pattern file needs to have an extra HI initialed line to specify the position in the input sequence at which the pattern occurrence of interest starts.

2. Setup

Installation of the standalone blast archive is fairly easy. Once the archive is placed in a desired directory and extracted, the whole package will be installed in a newly created subdirectory called blast-2.2.13 (assuming 2.2.13 release here). All the programs, including seedtop, will be in the blast-2.2.13/bin/ subdirectory (blast-2.2.13\bin\ for PC).

Appropriate setup requires the creation of .ncbirc configuration file, which blast programs (including seedtop) read upon startup to locate the appropriate files needed. In this .ncbirc, we can specify the location of the DATA directory and the BLASTDB directory using the following lines:

[NCBI]
DATA=/path/data

[BLAST]
BLASTDB=/path/db

The [NCBI] section is used by most of the NCBI programs to locate the data directory and retrieve specific files needed (MATRIX file for example). The [BLAST] section specifies the path to the directory where databases are stored.

The db directory does not come with the NCBI setup, so one needs to create it after installation. If we place the directory anywhere, we need to change the path correspondingly. For simplicity, we suggest that it be created under the blast-2.2.13 (version # will vary for future releases) at the same level as data directory.

3. Program Options

Once the standalone BLAST package is setup, we can "cd" to the directory and issue the "seedtp -" command to display the program options for this program. Here we list each option in a table and describe its functions, argument value, and example usage.

Table 3.1
Option	-d
Function	Specifies the target database to search
Default	nr
Input format	Takes database formatted by formatdb, use name without extension
Example	To search against est_human, use: -d est_human
Note	This is not a mandatory option, search for patterns in a single input sequence does not require this option.

Table 3.2
Option	-i
Function	Specifies the input query file
Default	stdin
Input format	[File In], file name with extension
Example	To take my_pept.txt as input query, use: -i my_pept.txt
Note	To using stdin as input, either redirect or pipe the input: seedtop -k pat -p patmatchp <input_file more input_file\| seedtop -k pat -p patmatchp

Table 3.3
Option	-k
Function	Specifies the input pattern (Hit File)
Default	hit_file
Input format	Complete file name with extension
Example	If the pattern file is named my_pat.txt, use: -k my_pat.txt
Note	See section 4.1 below for details.

Table 3.4
Option	-o
Function	Specifies the output file name
Default	stdout
Input format	file name with or without extension
Example	To save result in my_output, use: -o my_output
Note	Redirection or piping can be used instead.

Table 3.5
Option	-G
Function	Specifies the cost to open a gap
Default	11
Input format	[Integer]
Example	To change this to 12, use: -G 12
Note	The choice of -M option determines the available input value for this option as well as that for -E option. Only a selected set is supported. Detailed list is in the blastall document.

Table 3.6
Option	-E
Function	Specifies the cost to extend a gap
Default	1
Input format	[Integer]
Example	To change this to 2, use: -E 2
Note	See Table 3.5 for more information

Table 3.6
Option	-D
Function	Specifies the cost to decline alignment
Default	99999
Input format	[Integer]
Example	N/A
Note	Functions similar to the -L option in blastpgp. If enabled, it would implement Dr. Altschul's 3-parameter gap model for scoring.

Table 3.7
Option	-X
Function	Specifies X dropoff value for gapped alignment (in bits)
Default	15
Input format	[Integer]
Example	To increase this dropoff value to 20, use: -X 20
Note	Increasing this value may enable one to see a longer alignment

Table 3.8
Option	-S
Function	Specifies cutoff cost
Default	30
Input format	[Integer]
Example	N/A
Note	Currently it is overridden in pseed3.c. It could allow the user to control the score threshold applied to the part of the alignment that does not include the pattern in deciding which alignment(s) to report.

Table 3.9
Option	-C
Function	Score only or not
Default	1
Input format	[Integer]
Example	N/A
Note	This is relevant only to searches with -p seed(p) or -p patseed(p). NOT implemented yet.

Table 3.10
Option	-I
Function	Whether to Show GI's in deflines
Default	F
Input format	[T/F]
Example	To display GI in the deflines, use: -I T
Note	Relevant only to searches with -p seed(p) or -p patseed(p)

Table 3.11
Option	-e
Function	Specifies the expectation value (E) cutoff
Default	10.0
Input format	[Real]
Example	To set this to 0.001, use: -e 0.001 or -e 1e-3
Note	Relevant only to searches with -p seed(p) or -p patseed(p)

Table 3.12
Option	-J
Function	Whether to believe the query defline or not
Default	F
Input format	[T/F]
Example	To set this to true, use: -J T
Note	To save SeqAlign object requires -J T

Table 3.13
Option	-O
Function	Specifies the output file for SeqAlign object
Default	Optional
Input format	[File Out]
Example	N/A
Note	Relevant only to searches with -p seed(p) or -p patseed(p). NOT implement yet.

Table 3.14
Option	-M
Function	Specifies which matrix file to use
Default	BLOSUM62
Input format	[String]
Example	To set matrix to PAM30, use: -M PAM30
Note	Relevant to seedp/patseedp searches, only a limited set is supported

Table 3.15
Option	-p
Function	Specifies which subprogram to run
Default	patmatchp
Input format	[String]
Example	To find protein patterns in a database, use: -p patternp
Note	Choices for nucleotide searches: patmatch, pattern, seed, and patseed Choices for protein searches: patmatchp, patternp, seedp, and patseedp

Table 3.16
Option	-r
Function	Specifies the reward for a match
Default	10
Input format	[Integer]
Example	To increase the reward to 20, use: -r 20
Note	Relevant only to searches with -p seed(p) or -p patseed For nucleotide searches only.

Table 3.17
Option	-q
Function	Specifies the cost for a mismatch
Default	-10
Input format	[Integer]
Example	To increase the penalty to -15, use: -q -15
Note	For nucleotide search with seed/patseed only.

Table 3.18
Option	-F
Function	Whether to filter query sequence with SEG
Default	F
Input format	[T/F]
Example	To activate filter, use: -F T
Note	Relevant to seedp/patseedp searches only.

4. Execution and Practical Usage

The most useful functionalities of seedtop are patmatchp and patternp. Since patseedp does not generate the actual alignment and its function is already incorporated in blastpgp, we will not cover it here. The functionality for searching with nucleotide entries are similar to protein searches, we will only provide a couple simple examples.

4.1 Pattern specification

The pattern input file is unique for seedtop. Each pattern is specified by two lines, ID initialed for identification and PA initialed for pattern. The pattern specification uses the ProSite syntax. Multiple patterns can be used, each separated by a single line with a "/".

Single pattern specification:

ID Cyclic nucleotide-binding domain signature 2.
PA [LIVMF]-G-E-x-[GAS]-[LIVM]-x(5,11)-R-[STAQ]-A-x-[LIVMA]-x-[STACV].

Multiple pattern specification:

ID Cyclic nucleotide-binding domain signature 2.
PA [LIVMF]-G-E-x-[GAS]-[LIVM]-x(5,11)-R-[STAQ]-A-x-[LIVMA]-x-[STACV].
/
ID Cyclic nucleotide-binding domain signature 1
PA [LIVM]-[VIC]-x-{H}-G-[DENQTA]-x-[GAC]-{L}-x-[LIVMFY](4)-x(2)-G

Pattern lines should be less than 100 letters long. Longer patterns can be broken up into two or more PA lines. Multiple individual patterns should be separated by a line containing a single backslash (/). Other general pattern rules can be summarized as the following:

Symbol Meaning

[] marks a single position, match to anyone in the bracket is acceptable

(x,y) marks a range for the residue(s) before it, matching within the range is acceptable

(x,) represents range with no upper limit for the residue(s) before it

(x) represents exact number of matches for the residue(s) before it

{} marks a single residue, residues in the braces should be excluded

- separates the individual positions in the pattern

. used at the end marks the end of a pattern

> symbol at the end marks an incomplete pattern (optional)

4.2 patmatchp

This function matches patterns found in an input pattern file and identifies the pattern occurrences in an input protein sequence. The sample command line below (given along with its output) takes an input pattern named pattern.txt, searches against the input query.aa target sequence, and saves the output in a file named query.out.

seedtop -k pattern.txt -i query.aa -p patmatchp -o query.out

Name Cyclic nucleotide-binding domain signature 2.
Pattern [LIVMF]-G-E-x-[GAS]-[LIVM]-x(5,11)-R-[STAQ]-A-x-[LIVMA]-x-[STACV].
At position 521 of query sequence
Name Cyclic nucleotide-binding domain signature 1
Pattern [LIVM]-[VIC]-x-{H}-G-[DENQTA]-x-[GAC]-{L}-x-[LIVMFY](4)-x(2)-G
At position 483 of query sequence

The result only lists the pattern starting positions in the query sequence.

4.3 patternp

This function matches patterns in an input pattern file against an input database and reports back the database entries containing one or more of the input patterns as well as the pattern locations. The sample command line below (given with its partial output) takes an input pattern named pattern.txt, searches against the input refseq_protein database, the identified entries with pattern matches are saved in the output file db.out.

seedtop -k pattern.txt -d refseq_protein -p patternp -o db.out

seqno=892602 gi|33859524|ref|NP_034048.1|

ID Cyclic nucleotide-binding domain signature 1
PA [LIVM]-[VIC]-x-{H}-G-[DENQTA]-x-[GAC]-{L}-x-[LIVMFY](4)-x(2)-G
HI (449 450) (452 454) (456 457) (459 462) (465 465)
seqno=892873 gi|51470807|ref|XP_290552.4|

ID Cyclic nucleotide-binding domain signature 1
PA [LIVM]-[VIC]-x-{H}-G-[DENQTA]-x-[GAC]-{L}-x-[LIVMFY](4)-x(2)-G
HI (374 375) (377 379) (381 382) (384 387) (390 390)

Each hit is described in the following lines:

seqno: seqid for the database sequence with pattern matches
ID: Pattern ID, reiterated pattern input
PA: Pattern, reiterated pattern input
HI: Hit position on the db sequence, regions broken up by X

4.4 patseedp

This function takes three inputs, an input pattern, a query protein sequence with the pattern, and a protein sequence database. It identifies the pattern in the query and aligns the query against the database entries that contains the same pattern. It reports the pattern position in the query, the total number of pattern occurrences in the database, and the actual database entries with pattern and alignment to the input query. Specifically, it reports the seqid of the database entry, its alignment (with the query) E-value, scores, and pattern position.

seedtop -k pattern.txt -d refseq_protein -p patseedp -o pat_db.out -i query_aa.txt

1 occurrence(s) of pattern in query

Name Cyclic nucleotide-binding domain signature 2.
Pattern [LIVMF]-G-E-x-[GAS]-[LIVM]-x(5,11)-R-[STAQ]-A-x-[LIVMA]-x-[STACV].
At position 488 of query sequence
effective database length=3.1e+008
pattern probability=3.4e-008
lengthXprobability=1.0e+001

Number of occurrences of pattern in the database is 265
892602 gi|33859524|ref|NP_034048.1|
0 Total Score 3279 Outside Pattern Score 3162 Match start in db seq 488
Extent in query seq 1 631 Extent in db seq 1 631

1 occurrence(s) of pattern in query

Name Cyclic nucleotide-binding domain signature 1
Pattern [LIVM]-[VIC]-x-{H}-G-[DENQTA]-x-[GAC]-{L}-x-[LIVMFY](4)-x(2)-G
At position 450 of query sequence
effective database length=3.1e+008
pattern probability=7.0e-008
lengthXprobability=2.2e+001

Number of occurrences of pattern in the database is 247
892602 gi|33859524|ref|NP_034048.1|
0 Total Score 3279 Outside Pattern Score 3188 Match start in db seq 450
Extent in query seq 1 631 Extent in db seq 1 631

The input pattern.txt file contains two patterns, so the result contains two sections, one for each pattern. Only one database match is shown.

4.5. seedp

This function is similar to patseedp. The only difference is that the pattern file should specify the pattern position in the input query sequence. The output from the patternp can be used for this purpose. This specifies which pattern is to be used during the search.

An actual command line and pattern file is listed below. Output is omitted since it is essential the same as that from patseedp given above.

seedtop -p seedp -k pattern2a.txt -d refseq_protein -i q_aa.txt -o seed.out

ID Cyclic nucleotide-binding domain signature 1
PA [LIVM]-[VIC]-x-{H}-G-[DENQTA]-x-[GAC]-{L}-x-[LIVMFY](4)-x(2)-G
HI (450 451) (453 455) (457 458) (460 463) (466 466)

The seedp functionality has been incorporated into standalone blastpgp, on the BLAST web server, it is called Pattern-Hit-Initiated BLAST or PHI-BLAST. The differences are that blastpgp does not report the total number of pattern occurrences in the database, but it does generate actual sequence alignments. The implementation in blastpgp provides more functionality in that the results of the (first) round of PHI-BLAST search can be used seamlessly as the start of a PSI-BLAST iterated search.

4.6. For nucleotide searches

Search for nucleotide patterns using seedtop is very similar to what described above for proteins queries. The function names for nucleotide have no terminating p.

5. Technical Support

For additional questions and comments, please write to:

blast-help@ncbi.nlm.nih.gov

Questions and inquries on other NCBI resources should be sent to:

info@ncbi.nlm.nih.gov