SSR mining and primer design (MISA and Primer3)
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2.3 years ago
putita.jira ▴ 10

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

microsatellite SSR primer design MISA Primer3 • 2.0k views
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2.3 years ago
h.mon 33k

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.

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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?

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Do your contig names contain spaces or other special characters?

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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

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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


Removing space form fasta file header

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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.