Q1: Can I still use the primer pair designed by GetPrimers when warning messages are triggered?

A1: Since the location of G3 and G4 are more or less fixed by design, so there is not much room for primer optimization. Therefore, in some cases, warnings could be raised when the calculated G3 and G4 primer pair suffer from issues such as potential hairpin formation and suboptimal annealing temperature. In such cases, users are advised to make further adjustment on GetPrimers-designed primers themselves or the corresponding PCR reaction condition to work around this issue. For example, at least for gene knockout, G3 and G4 do not have to be at the exact boundary of start and stop codons. So their position could be manually shifted upstream or downstream by users afterwards when needed.

Q2: Why the genomic coordinate of CDS_start is less than that of CDS_end for genes on the negative strand in GetPrimers’ outputs?

A2: By design, the reported genomic coordinate is always based on the positive strand, in concordance with the original genomic coordinates in standard GFF annotation files. Luckily, GetPrimers output strand information. Therefore, when the strand value is “-”, the reported start and end coordinates of the corresponding CDS should be swapped to reflect the actual CDS start and end along its transcription direction.

Q3: How do I tell if my query gene ID or gene name is supported?

A3: Users can download the supported gene ID and gene name information from https://www.evomicslab.org/app/getprimers/geneid/.

Q4: How to prepare customized genome, annotation, and plasmid files for GetPrimers?

A4: We recommend downloading genome sequence (in FASTA format) and the corresponding annotation file (in GFF format) from the same source, preferably either from NCBI (https://www.ncbi.nlm.nih.gov/assembly/) or Ensembl (http://www.ensembl.org/ or http://fungi.ensembl.org/). If genome and annotation files are downloaded from other databases, then please make sure that these two files match with each other. For example the chromosome ID in the genome file should keep concordance with that in the annotation file. In the annotation file, the annotated genomic features follow the order of gene -> mRNA -> CDS. Regarding the plasmid file, users only need to provide the 5’->3’ sequence of the gene targeting cassette, not the whole plasmid sequence.

Q5: What can I do if I want to design targeting primers with a newer version of the genome/annotation build for my favorite organisms while the the pre-shipped genome/annotation versions in GetPrimers appears out-of-date?

A5: we will check and update the incorporated genome and annotation files every year to keep them up to date, especially for classic yeast models such as Saccharomyces cerevisae and Schizosaccharomyces pombe. Users are also welcome to contact us directly to raise the request.

Q6: How does GetPrimers deal with genes with multiple versions of transcripts?

A6: If a gene has multiple versions of transcripts and the feature 'mRNA' has been defined in the annotation GFF3 file, GetPrimers use the first transcript appearing in the annotation GFF3 file. If the feature 'mRNA' is missing in the annotation GFF3 file, GetPrimers will use the outermost CDSs to define the coding region boundaries for its primer design.

Q7: How to design gene targeting primers for a specific locus that is not explicitly annotated in GFF file?

A7: Users can provide a customized GFF file that only contain the interested locus and then upload it together with the associated genome file to GetPrimers' webserver for primer design. Note that the locus should be defined as a gene locus and the chromosome ID should match the chromosome ID defined in the genome file. An example of such customized locus-specific GFF file can be defined as follows:

chr01	Custom	gene	2000	3000	.	+	.	ID=gene-custom1
chr01	Custom	mRNA	2000	3000	.	+	.	ID=mRNA-custom1
chr01	Custom	CDS	2000	3000	.	+	.	ID=cds-custom1

Q8: Why my uploaded custom genome take very long to process?

A8: When designing primers for a custom organism with >20 Mbp genome size, we recommend to either contact us to add your genome or to install and run GetPrimers locally.