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General Guidance
PRIMEGENS can run from Unix/Linux shell as a stand-alone package. It has been tested extensively
on Sun, DEC, and Linux PC. Running PRIMEGENS from a Unix shell (rather
than from the Web interface) provides a convenient way to handle multiple
data sets and multiple runs automatically by using scripts.
All the options for the program can be specified through flags on
the command line. Multiple flags without conflicts can be used at
the same time, and their orders in the command line do not matter.
Default Run
To run primegens, you need a file (say "database") which records all the
known ORFs for the genome in the FASTA format.
You can simply run the program using default parameters:
primegens database > log
Output Files
- Log file ("log") which gives sequence alignments;
- Unique sequence files:
database.nohit (unique by itself)
database.seg (unique portion in the sequence)
database.sim (redundant sequences in the database)
- Primer information for each group:
database.nohit.primer (unique by itself)
database.seg.primer (unique portion in the sequence)
database.sim.primer (redundant sequences in the database)
- Input for Excel spread sheet (separated by tab):
database_primer.xl
The columns represent: plate number, row number, column number, ORF
name, gene function, forward primer, starting position of forward primer,
ending position of forward primer, melting temperature of forward primer,
reverse primer, starting position of reverse primer, ending position of
reverse primer, melting temperature of reverse primer, expected length
(the length between forward primer and reverse primer), length
of the gene, and length of the unique segment, respectively.
- The primer files with each having 96 primers:
primer_plate_left1 primer_plate_left2 ...
primer_plate_right1 primer_plate_right2 ...
Command Line Options
The following optional flags are available:
- Calculate primers on a subset of ORFs (genes) in "database".
primegens -l subset database
(if the file "subset" is in a list format, with each ORF in a line).
primegens -lf subset database
(if the file "subset" is in FASTA format).
- If ORFs in "database" has no functions defined, one can use
a separate function file (e.g., "function") in a format:
ORF1 function a
ORF2 function b
...
Then run
primegens -name function -l subset database
- Keep the BLAST outputs in the temporary directory (default: no):
primegens -bf database
- Define the maximum expectation value (default: 1e-15) to get a BLAST
alignment, e.g.,
primegens -e 1e-20 database
- Define the minimum length (default: 100 bases) to select a unique
segment, e.g.,
primegens -mseg 80 database
- Define the maximum sequence similarity (default: 75%) between the
select segment and any other sequence, e.g.,
primegens -msim 0.80 database
- Define the minimum length (default: 50 bases) to filter out a BLAST
alignment as a potential primer, e.g.,
primegens -mfil 30 database
- Define the faction (default: 0.8) of the length as the lower limit
in the "PRIMER_PRODUCT_SIZE_RANGE" parameter for Primer3, e.g.,
primegens -f 0.6 database
- Calculate the unique sequences only without designing primers
using Primer3:
primegens -np database
- Define the maximum length (-maxsep, default: 10000 bases) or
the minimun length (-minsep, default: 0) for the primer
pruduct, e.g.,
primegens -maxsep 1200 -minsep 100 database
You can combine several options together. The order of options does not
matter as long as the "database" file is at the end of the argument for
primegens. You can go to the "test" directory of the package and do a
test run:
primegens -msim 0.80 -name protein.list -l list ORFDNA > log
Editing the Conditions for Primers
If you like to change the conditions for generating primers, you also need copy the file
"append" in the primegens directory (e.g., /usr/local/primegens/) to the
directory you want to run the program, and modify the parameters in
"append" accordingly. Otherwise the default pa
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