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GetPrimers web application manual

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codeatcg edited this page May 3, 2022 · 5 revisions

Introduction

GetPrimers web application (https://www.evomicslab.org/app/getprimers/) was built with Django and bootstrap. The application shipped with a pre-built database for several strains. For strains 972h, OY26, yFS275, yFS286 and S288C genome and GFF files were downloaded from NCBI and Ensemble. Primers were designed separately for the two types of data by GetPrimers command line program. For strains DBVPG6044, DBVPG6765, SK1, Y12, YPS128, CBS432 and YPS138 genome and GFF files were from the recent assemblies and annotations, which were the update versions of Yeast Population Reference Panel (https://yjx1217.github.io/Yeast_PacBio_2016/data/). Both short primer strategy and long primer strategy were used. By default parameters and pre-built database, the web application is fast, but if the default parameters are changed the pre-built database will be neglected and primers will be redesigned and it may take several minutes if too many genes are submitted simultaneously. If you have many genes for which primers need to be redesigned and you have a computing server it’s better to run the command line program, parallelly.

Pre-built database
species strain
Schizosaccharomyces_pombe 972h
Schizosaccharomyces_cryophilus OY26
Schizosaccharomyces_japonicus yFS275
Schizosaccharomyces_octosporus yFS286
Saccharomyces_cerevisiae S288C, DBVPG6044, DBVPG6765, SK1, Y12, YPS128
Saccharomyces_paradoxus CBS432, YPS138

Home page

Home page of web site

Notes

Annotation GFF style

For stains 972h, OY26, yFS275, yFS286 and S288C NCBI and Ensembl are optional. For strains DBVPG6044, DBVPG6765, SK1, Y12, YPS128, CBS432 and YPS138 this option is invalid and can be neglected.

Upload assembly and Upload GFF

If you want to design gene targeting primers using your own assembly and annotation these two files are needed to be uploaded simultaneously.

Choose a pre-built plasmid cassette and Primer type

Please choose the plasmid cassette and primer type carefully. For example, a C’ tagging plasmid and N’ tagging primer type will cause incorrect results.

Gene list
Can be a single gene or a gene list. Gene name, gene ID and Dbxref ID are all supported for NCBI GFF. Dbxref ID are not supported for Ensembl GFF.

  • Annotation ID List:

    YAL067C
    YAL064C-A

  • Gene Name List:

    SEO1
    TDA8

  • Dbxref ID List:

    851230
    851234

Primer location

Commonly, section of G3, G4, P1 and P2 that are homologous to plasmid is not needed to be redesigned. The length of the section can be assigned. By default, this value is 20. If you want to redesign this section by melt temperature, bases composition and other criteria to design primers please select the check box ‘redesign primer common sequence, which is homologous to insertion cassette’ in ‘Other options’. By this option V5 and V6 are also redesigned.

Verification primers

By default, verification primers V5 and V6 are complementary to section of G3, G4, P1 and P2 that are homologous to plasmid.

Schematic and output information Please read https://www.evomicslab.org/app/getprimers/info/

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