THIS IS STILL IN DEVELOPMENT.
The Primer Design terminal program produces thermodynamically stable primer pairs for qPCR from an input of aligned nucleotide sequences. This is the main subcommand known as select. A preliminary subcommand is screen which produces a graph of the coverage and the entropy for each position of the input reference sequences. The input nucleotide sequences should be in the FASTA format.
Follow the instruction of (https://github.com/rdpstaff/RDPTools) to setup RDPTools. This will download PrimerDesign code and setup the ReadSeq dependency.
Then, the user is required configure the config file which is located in the PrimerDesign folder. It will require the java JNI include directory and operating system specific JNI directory. You will find these directories in your JAVA_HOME directory. On mac operating system, you can find the java_home using the command below:
/usr/libexec/java_homeYou can find java_home on linux using the following command:
dirname $(dirname $(readlink -f $(which javac)))The example config file has the java_jni_include and java_jni_include_os directories for my computer. The java_home directory is /Library/Java/JavaVirtualMachines/jdk1.8.0_121.jdk/Contents/Home/ You will find the include directory in java_home in my case it is /Library/Java/JavaVirtualMachines/jdk1.8.0_121.jdk/Contents/Home/include/. In the JNI include directory, you will find operating system specific folder in my case it is /Library/Java/JavaVirtualMachines/jdk1.8.0_121.jdk/Contents/Home/include/darwin. These directories are required to compile primer3 native code.
You can build the PrimerDesign jar using the ant command:
ant -f PrimerDesign/build.xml jarGo to the PrimerDesign directory and compile the primer3 native code using the command below:
makeAfter you run the above commands, you can find the PrimerDesign.jar in the PrimerDesign/dist folder.
java -Xmx8g -jar /path/to/PrimerDesign.jar [--help]
--assayMax <assayMax> assayMax (default:
20) - Maxiumum
number of assays
allowed - one
degenerate primer
pair per assay
--degenMax <degenMax> degenMax (default:
8) - Maximum
degeneracy per
primer pair.
Nondegenerate
primers = 1.
Recommend no
higher than 10
--forwardMaxPos <forwardMaxPos> forwardMaxPos
(default: 120) -
Foward oligo
maximum position
to end
enumeration.
Needed if
SlidingScale is
false
--forwardMinPos <forwardMinPos> forwardMinPos
(default: 100) -
Foward oligo
minimum position
to begin
enumeration.
Needed if
SlidingScale is
false
--GCFilterMax <GCFilterMax> GCFilterMax
(default: 0.70) -
G+C content filter
maximum percent.
Recommend leaving
at default if
unsure
--GCFilterMin <GCFilterMin> GCFilterMin
(default: 0.45) -
G+C content filter
minimum percent.
Recommend leaving
at default if
unsure -h,--help Shows this help
--hairMax <hairMax> hairMax (default:
35) - Hairpin
maximum
temperature
--homoMax <homoMax> homoMax (default:
35) - Homodimer
maximum
temperature
-i,--input <input> Input .fasta or
.fa file. Contains
aligned gene
sequences
--isHenikoffWeightNeeded <isHenikoffWeightNeeded>
isHenikoffWeightNe
eded (default:
false) - Henikoff
Weighting Method.
Set to 'true' or
't' if you would
like this
weighting method
results aswell as
uniform results.
Will give the
highest weight to
unique sequences
based on bases at
each position.
--isTreeWeightNeeded <isTreeWeightNeeded> isTreeWeightNeeded
(default: false)-
A phylogenetic
tree weighting
method. Must
include .tree
file. Set to
'true' or 't' if
you would like
this weighting
method results
aswell as uniform
results. Will give
the highest weight
to sequences
closest to the
root
--magnesConc <magnesConc> magnesConc
(default: 1.5) -
Divalent magnesium
concentration for
thermodynamic
calculations
--maxMismatches <maxMismatches> maxMismatches
(default: 2) -
Oligo mismatch
maximum. Recommend
setting between 0
and 3
--NoPoly3GCFilter <NoPoly3GCFilter> NoPoly3GCFilter
(default: true) -
If true, filter to
remove any oligo
generated with
three Guanines or
three Cytosines in
a row
--NoTEndFilter <NoTEndFilter> NoTEndFilter
(default: true) -
If true, filter to
remove any oligo
generated with a
Thymine base at
end
-o,--GraphOutput <GraphOutput> If screen
subcommand is ran,
then this is the
output directory
for graph results
--oligoMaxSize <oligoMaxSize> oligoMaxSize
(default: 30) -
Maximum oligo
size; Recommend no
longer than 30 bp
--oligoMinSize <oligoMinSize> oligoMinSize
(default: 22) -
Minimum oligo
size; Recommend no
shoter than 15 bp
--os <os> Operating System :
linux/mac
(default: mac)
--output <output> Full path to
output file (for
select)
--PolyRunFilter <PolyRunFilter> PolyRunFilter
(default: 4) -
Poly Run max
filter
--productLengthMax <productLengthMax> productLengthMax
(default: 200) -
Maximum amplicon
product length.
Needed if
SlidingScale is
true
--productLengthMin <productLengthMin> productLengthMin
(default: 100) -
Minimum amplicon
product length.
Needed if
SlidingScale is
true
--reverseMaxPos <reverseMaxPos> reverseMaxPos
(default: 370) -
Reverse oligo
maximum position
to end
enumeration.
Needed if
SlidingScale is
false
--reverseMinPos <reverseMinPos> reverseMinPos
(default: 350) -
Reverse oligo
minimum position
to begin
enumeration.
Needed if
SlidingScale is
false
--SlidingScale <SlidingScale> SlidingScale
(default: true) -
If true, then
forward and
reverse primer
pairs are built
with a sliding
window between the
given amplicon
product minimum
and maximum
length. If false,
the pairs will be
built between the
forward and
reverse given min
and max.
--sodiumConc <sodiumConc> sodiumConc
(default: 50) -
Monovalent sodium
concentration for
thermodynamic
calculations
--subcommand <subcommand> select subcommand:
select - file
output of
individual
degenerate primer
pairs with
sequences
theoretically hit
/ screen - graph
output of the
proportional
theoretical
uniform coverage
at each position
(default: select)
-t,--treeinput <treeinput> File (.tree)
containing
phylogentic
relationship of
the input
sequences. In
Newick format.
Must have if tag
-isTreeWeightNeede
d is true
--tempMax <tempMax> tempMax (default:
63) - Oligo max
melting
temperature
--tempMin <tempMin> tempMin (default:
55) - Oligo min
melting
temperature
RDP Primer Design Subcommands: Screen and Select
Screen – command used to generate a graphical representation of the coverage and entropy at each position of the reference set. A proportional bar graph of the theoretical uniform coverage is calculated at any given position as the summation of all possible targets hit divided by the total number of sequences.
Sample Screen Command
java -jar /path/to/PrimerDesign.jar -subcommand screen -input /path/to/refSequences.fasta -GraphOutput /path/to/userDir -oligoMinSize 15 -oligoMaxSize 30 -tempMin 55 -tempMax 63 -hairMax 35 -homoMax 35 -os mac -NoTEndFilter t -NoPoly3GCFilter t -PolyRunFilter 4 -GCFilterMin 0.45 -GCFilterMax 0.7 -assayMax 30 -degenMax 6 -sodiumConc 50.0 -magnesConc 1.5Select – command that generates an output file of primer pairs with its corresponding target sequences. Initial program settings given by the user for the user’s record are also given as output.
A user should consider the benefits of the insight from the screen results when running select.
Perhaps there are high peaks of coverage slightly outside of the user’s initial desired amplicon product minimum and maximum lengths. The tag ‘IsSlidingScale’ can be set to ‘false’ to stop the generation of primer pairs from all positions with a sliding window. Then, set the forward and reverse minimum and maximum to position ranges that have high coverage and relatively moderate to low richness.
Sample Select Command (without Sliding Scale)
java -jar /path/to/PrimerDesign.jar -subcommand select -input /path/to/refSequences.fasta -SlidingScale false -forwardMinPos 100 -forwardMaxPos 150 - reverseMinPos 425 -reverseMaxPos 450 -oligoMinSize 15 -oligoMaxSize 30 -maxMismatches 2 -tempMin 55 -tempMax 63 -hairMax 35 -homoMax 35 -isTreeWeightNeeded f -isHenikoffWeightNeeded t -os mac -output /work/leotift/nifHGrp2CmdLineResults -assayMax 30 -degenMax 6 -NoTEndFilter t -NoPoly3GCFilter t -PolyRunFilter 4 -GCFilterMin 0.45 -GCFilterMax 0.7 -sodiumConc 50.0 -magnesConc 1.5Perhaps coverage does not vary significantly based upon position. This would be the time to just use the sliding window with a product length minimum and maximum.
Sample Select Command (with Sliding Scale)
java -jar /path/to/PrimerDesign.jar -subcommand select -input /path/to/refSequences.fasta -SlidingScale true -productLengthMin 150 -productLengthMax 250 -oligoMinSize 15 -oligoMaxSize 30 -maxMismatches 2 -tempMin 55 -tempMax 63 -hairMax 35 -homoMax 35 -isTreeWeightNeeded f -isHenikoffWeightNeeded t -os mac -output /work/leotift/CmdLineResults -assayMax 30 -degenMax 6 -NoTEndFilter t -NoPoly3GCFilter t -PolyRunFilter 4 -GCFilterMin 0.45 -GCFilterMax 0.7 -sodiumConc 50.0 -magnesConc 1.5Sample Output File of the Select subcommand (Input parameters, degenerate pairs with foward and reverse oligos, and sequences hit by each pair):
Michigan State University: RDP Primer Design
--------------------------------------------
Program User Settings
--------------------------------------------
Fasta file: /work/leotift/nifDgr3.fasta
Sliding Scale - t or f? false
Tree Weighted - t or f? false
Henikoff Weighted - t or f? false
Forward Start Position: 840
Forward End Position: 900
Reverse Start Position: 1130
Reverse End Position: 1170
Filters: T end: true, No Poly3GC: true, Poly Run: 4, GC Content:0.35 - 0.6
Oligo Min Size: 17
Oligo Max Size: 28
Max Number Mismatches: 0
Melting Temp Min: 55.0
Melting Temp Max: 62.0
Homodimer Max: 35.0
Hairpin Max: 35.0
Sodium Conc: 50.0
Magnesium Conc: 1.5
Max Degeneracy for each primer: 8
Max Num of Assays: 30
Sequence Length: 2343
--------------------------------------------
Primer Pair: DegeneratePair{fwdOligo=[Oligo{seq=ATCTCAATCTGCAGGTCACCA}{ Tm:58.65179472145502}{EndPos:899}
, Oligo{seq=CAATCAGCCGGTCACCA}{ Tm:56.34783911091415}{EndPos:899}
, Oligo{seq=GTAAGTCAATCAGCAGGACACCA}{ Tm:60.12327281754136}{EndPos:899}
, Oligo{seq=GAGTAACTCAATCAGCAGGTCATCA}{ Tm:60.18887096375721}{EndPos:899}
, Oligo{seq=AGCCAATCTGCAGGTCATCA}{ Tm:58.94355676316599}{EndPos:899}
, Oligo{seq=TATCTCAATCAGCAGGACATCA}{ Tm:56.262480666380384}{EndPos:899}
, Oligo{seq=TATCTCAATCAGCAGGGCACCA}{ Tm:61.0491700371382}{EndPos:899}
, Oligo{seq=CCCAATCTGCAGGACATCA}{ Tm:56.68496896549226}{EndPos:899}
] revOligo=[Oligo{seq=GAATAACATTCAAATCAGCAGTGTG}{ Tm:57.072928924899145}{EndPos:1140}
, Oligo{seq=ACTAGGTTTAAGTCTGCTTTGTC}{ Tm:56.05430626064259}{EndPos:1140}
, Oligo{seq=ACCAAGTTAAGTTCTGCAGTATG}{ Tm:56.37016198994928}{EndPos:1140}
, Oligo{seq=CCATGTTAAGGTCGGCTGTATG}{ Tm:58.703122878983265}{EndPos:1140}
, Oligo{seq=GGACTAGATTTAAATCTGCAGTATG}{ Tm:55.190491728626284}{EndPos:1140}
, Oligo{seq=GTACTAAGTTTAAATCCGCTGTATC}{ Tm:55.47307144157952}{EndPos:1140}
, Oligo{seq=TGCACTAAGTTTACATCTGCAGTGTG}{ Tm:61.5064061970719}{EndPos:1140}
, Oligo{seq=CCAAATTTACGTCTGCCTGATA}{ Tm:56.08567303175715}{EndPos:1140}
]}
Sequences hit
CP000721
CP002660
AE001437
CP016280
CP002118
AKVM01000053
LBCX01000114
LROS01000077
AKVN02000001
CP006763
CP010086
AKVJ01000024
CP006777
CBXI010000026
ABDT01000049
LBCY01000198
JPGY01000007
AF266462
JPGY02000001
CP011966
AYXR01000045
ANZB01000018
MBAF01000026
CP012395
ACOM01000001
CP014170
AY603957
AZLX01000216
LBCZ01000223
CP009268
CP009267
AY649324
AKVN01000021
LBCW01000051
AKVK01000143
AKVL01000569