-
Notifications
You must be signed in to change notification settings - Fork 12
/
getkasp.xml.example
executable file
·169 lines (100 loc) · 8.29 KB
/
getkasp.xml.example
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
<tool id="snpmarker" name="SNP Primer Design">
<description>for each sequence in a CSV file</description>
<command interpreter="python2">run_getkasp.py $input $ploidy $price $CAPS $KASP 0 $maxTm $maxSize $pick_anyway $reference</command>
<inputs>
<param format="txt" name="input" type="data" label="CSV file"/>
<param name="reference" type="select" label="Which reference to use?">
<option value="/Library/WebServer/Documents/blast/db/nucleotide/161010_Chinese_Spring_v1.0_pseudomolecules.fasta">Chinese_Spring_RefSeq_v1.0</option>
<option value="/Library/WebServer/Documents/blast/db/nucleotide/IWGSC_v2_ChrU.fa">IWGSC_v2_ChrU</option>
</param>
<param name="ploidy" size="4" type="integer" value="3" label="Ploidy" help="A genome, AB genome, or ABD genome"/>
<param name="price" size="4" type="integer" value="200" label="Maximum NEB restriction enzyme price (dollars per 1000 U)"/>
<param name="maxTm" size="100" type="text" value="63.0" label="Maximum Primer Tm"/>
<param name="maxSize" size="100" type="text" value="25" label="Maximum Primer Size (no larger than 36)"/>
<param name="pick_anyway" type="select" label="Pick primer anyway?" help="If yes, primers will be picked even if it violates specific constraints. Choose this only if you do not get any primers.">
<option value="0">No</option>
<option value="1">Yes</option>
</param>
<param name="CAPS" type="select" label="Design CAPS/dCAPS primers?" help="If yes, CAPS/dCAPS primers will be designed.">
<option value="1">Yes</option>
<option value="0">No</option>
</param>
<param name="KASP" type="select" label="Design KASP primers?" help="If yes, KASP primers will be designed.">
<option value="1">Yes</option>
<option value="0">No</option>
</param>
</inputs>
<outputs>
<data format="tabular" name="blast_out" from_work_dir="blast_out.txt" label="${tool.name} SNP sequence BLAST output"/>
<data format="tabular" name="caps_out" from_work_dir="Potential_CAPS_primers.tsv" label="${tool.name} CAPS primer output"/>
<data format="tabular" name="kasp_out" from_work_dir="Potential_KASP_primers.tsv" label="${tool.name} KASP primer output"/>
<data format="fasta" name="alignment_out" from_work_dir="All_alignment_raw.fa" label="${tool.name} Sequence Alignments"/>
</outputs>
<help>
**OVERVIEW**
This pipeline can make KASP primers and CAPS/dCAPS primers based on SNP flanking sequences.
The input file is the same as polymarker, e.g. a csv file with each line looks like this:
IWA7892,1BL,AGGATTCACGGGAAAAGATTTCGTCGCA[C/T]GTGCTAGGGTCTTTGCAGAATGTTA
------
**How SNP Primer Design works?**
1. Blast each SNP sequence against the pseudomolecule and get hits that are
**90% similarity and 90% of length of the best hit and > 50 bp**
2. Get 500 bps on each side of the SNP for all the hits
3. Multiple Sequence Alignment of the homeolog sequences with MUSCLE
4. Find all the variation sites that can differ the target from other homeologs
5. Use these sites as forced 3’ end in Primer3 and design homeolog specific sequences
6. Blast all the primers against the pseudomolecule v1.0 with word size 7 to see whether it also hits other chromosomes
**Criterion of matches: less than 2 mismatches in the first 4 bps from 3’**
------
**Restriction Enzymes**
Restriction Enzymes used in this tool are all from NEB. And the prices were obtained on 05/29/2017.
I processed these enzymes and enzymes with the same recognization sequences were merged together. For example, MspI and HpaII both recognize "ccgg", so I give them the name "MspI,HpaII,13"; the number in the end is the lowest price (US dolloars per 1000 U) of the enzyme group.
**FAM and VIC/HEX tails**
FAM GAAGGTGACCAAGTTCATGCT
VIC GAAGGTCGGAGTCAACGGATT
You should add FAM and VIC tails for the two allele specific primers.
------
**Output Format**
Four output files will be availabe after each run:
1. Multiple sequence alignment file for each marker
2. Blast output for each SNP
3. Primer pairs for KASP design, and
4. primer pairs for CAPS design
For the KASP and CAPS output, below the primer pairs for each marker are the positions that can differ all the other homeologs (Uniuqe to the template). These positions are used for 3' end of primers in Primer3. When selecting primers, if it has more than 1 of these positions in the first 4 bases from the 3', then it would be more specific to this chromosome.
Results of **primer blast** against the IWGSC pseudomolecule v1.0 was in the last column of the KASP and CAPS output file. The selection criterion is: less than 2 mismatches in the first 4 bases from the 3' of each primer.
------
**KASP Primers**
**index product_size type start end variation number 3'diffall length Tm GCcontent any 3' end_stability hairpin primer_seq ReverseComplement penalty compl_any compl_end PrimerID matched_chromosomes**
CSS7AL4506666-1053-371-0-G 169 RIGHT 520 501 0 YES 20 62.241 60 15.93 12.79 4.4 0 gacaggaacgcgaacatggC Gccatgttcgcgttcctgtc 3.136142 0 0 R1 ;chr7A:673419067
CSS7AL4506666-1053-371-0-C 169 RIGHT 520 501 0 YES 20 62.241 60 15.93 12.79 4.4 0 gacaggaacgcgaacatggG Cccatgttcgcgttcctgtc 3.136142 0 0 R1 ;chr7A:673419067
CSS7AL4506666-1053-371-0-Common 169 LEFT 352 371 3 YES 20 59.104 55 0 0 4.26 41.05 ccgaccaaagTaggagcTgA TcAgctcctActttggtcgg 3.136142 0 0 L10 ;chr7A:673418899
For the common primer, captial letters in the primer sequences are unique nt to the template, and the column "variation number" has the count of these nt, and the more the better. **However, because I shifted the alignment where there is an indel, sometimes this cause too many mismatches and "variation number" will be very big, and be cautious for these primers.**
chr7A:673418899 means it hits on chr7A at position 673418899
KASP primer product size is set from 50 bp to 250 bp, and usually the shorter the better.
------
**dCAPS primers**
**index product_size type start end diff_number length Tm GCcontent any 3' end_stability hairpin primer_seq ReverseComplement 3'differall penalty compl_any compl_end PrimerID matched_chromosomes**
CSS7AL4435040-1801-dCAPS-HhaI,HinP1I,29-gcgc-312-502-0 229 LEFT 292 312 1 21 60.134 47.619 0.0 0.0 3.09 32.98 accaaaggtgttcacggcttA Taagccgtgaacacctttggt YES 2.576185 0.00 0.00 L11 ;chr7A:674139835;chr5D:47531811;chr5A:37935864;chr7B:661719815
CSS7AL4435040-1801-dCAPS-HhaI,HinP1I,29-gcgc-312-502-0 229 RIGHT 520 502 0 19 59.558 63.158 20.69 0.0 4.85 43.94 gagatggtaccggtctgGc gCcagaccggtaccatctc YES 2.576185 0.00 0.00 R4
For dCAPS primers, primers with the introduced mutations have to be close to the SNP, so primers with end position close to 501 are the primers with introduced mutations (I got 500 bp each side of the SNP), and the other primer is the primer to insure the pair is chromosome specific. For the primers with introduced mutations, the captital letters mean the introduced mutation, and for the other primer, the capital letters are the unique nts.
dCAPS product size is set 70 bp to 350 bp, usually 100 bp to 200 bp is better. **One trick: add a FAM or VIC tail in the primer with the introduced mutation can make the difference about 40 bp**
**CAPS primers**
**index product_size type start end diff_number length Tm GCcontent any 3' end_stability hairpin primer_seq ReverseComplement 3'differall penalty compl_any compl_end PrimerID matched_chromosomes**
CSS7AL4506666-1053-CAPS-BsaJI,66-ccnngg-243-0 325 LEFT 224 243 9 20 59.746 55 0 0 2.75 30.59 acTGcacCatCcACCagaTC GAtctGGTgGatGgtgCAgt YES 0.290188 12.48 12.48 L1 ;chr7A:673418771
CSS7AL4506666-1053-CAPS-BsaJI,66-ccnngg-243-0 325 RIGHT 548 529 0 20 60.036 60 0 0 4.61 39.25 ctaggagcacaaggtgggtg cacccaccttgtgctcctag YES 0.290188 12.48 12.48 R1 ;chr7A:673419095
For CAPS primers, I only set the 1 of the primer 3' end as the unique nt in the template, and let Primer3 choose another one. The product size is set from 300 bp to 900 bp.
------
**How to select primers from the output**
1. Genomic specific:
-variation sites close to 3’ end
-more variation sites
-no blast hits on the other chromosomes
2. Good product size
-KASP: 50 – 250, the shorter, the better
-dCAPS: 70 – 350, I usually select 150-200
3. Low Tm difference: the smaller, the better
4. Low hairpin: the smaller, the better
------
For more information, please check: https://github.com/pinbo/SNP_Primer_Pipeline
</help>
</tool>