Hi everyone!
I'm trying to mine SSR from my assembled sequences and design primer pairs for them.
I used MISA for SSR mining. As the manual stated, I supposed to get 2 output files: in .misa format and .statistics format. However, what I got are a .statistics file and more than 100,000 .gff files! That made me shocked.
At first, I planned to use a .misa output for primer design in Primer3. But when I encounter with a ton of .gff files, I really don't know what to do next. I already searched it if anyone has the same problem but no, others seem to get a .misa file from MISA.
Can I use those .gff with Primer3? Do I have to input a .gff file at a time? (so if I want to design primer for all SSR, I have to run at least 100,000 times) Did anyone have the same problem as mine? How did you solve it? Or anyone else could run it successfully without a problem?
Thank you, Putita
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putita.jira •
You forgot to say you are using MISA 2.0 and you had GFF: true in your misa.ini file. If you don't want the gff files, edit the misa.ini to GFF: false.
The .misa output should be found along the gff files, try a ls *.misa to check. You can use the .misa output with the p3_in.pl script to prepare input for Primer3 - Primer3 doesn't take gff as input.
Thank you h.mon I really forgot to change gff setting in misa.ini file because I intended to use all default mining settings. Now I have a .misa file and I run this syntax
p3_in.pl FASTA.fa.misa
But I got an empty .p3in file. Could you help me figure out what might be wrong?
In .misa file, no. It looks like this.
ID SSR nr. SSR type SSR size start end
1_302_5934 1 p3 (TAT)7 21 1 21
1_302_5934 2 p3 (ATT)23 69 234 302
9_78_1277 1 c (TAA)5c(AAT)9aac(AAT)10 76 1 76
12_247_3182 1 c (CATC)16tattcatccattcatacatctatccatctatccacccg(TCCA)7 130 1 130
13_233_4645 1 c* (ATC)15(GTC)10* 75 1 75
But in contig file, there are spaces. By the way, this contig file could give normal .misa file.
>1 302 5934
TATTATTATTATTATTATTATTGTCGTTGCTGTTGTTATAGTTAGCACAGTAATATTATTGTTGTTATTATTATTAACATCGTTACAGGATAAGGCAGAATAGCAAGTGCTTTACAAATGTCATTTAAGCAAAAAAATACAAAGTAACTGAAGTACGTTGTGAGGTTTCAAGATTTGTGCCTTTCTGGTACCATTTGCTTTAATGACCGAGCTAAAAAAAACTGAGCTTTCCGATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATT
>4 99 209
TGGTATAAAAGAAAATGATGTCTAGAAACTTTTTTTCAATGATCCAGACCCGATGCAAATTTTCATTTCTTTTACATGGTCCCGTAACGAAGGAATCCC
>7 85 503
AAATATTCACATTGTAGCGCAGATATTTTTATTCAAGAAAATGTTCAATTAGCTAAAATGTGAGTGATTCAGCACACATGGTTAA
>9 78 1277
TAATAATAATAATAACAATAATAATAATAATAATAATAATAATAACAATAATAATAATAATAATAATAATAATAATAA
Replace the spaces at the headers of the fasta file by underscores and then try p3_in.pl again.
There are many threads about modifying fasta files, this one ( A: How to remove space in headers of fasta files ) has a nice and easy solution using BBTools:
reformat.sh in=reads.fasta out=fixed.fasta addunderscore
Some other threads:
Thank you so much h.mon for helping me.
I replaced space with underscore in config file and it worked well!
I ran the syntax until the last one which supposes to generate primer results in tabular format.
p3_out.pl FASTA.fa.p3out FASTA.fa.misa
but it was error. I found here that it has to do with version of Primer3. I'm trying to solve it.
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