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Primaclade FAQ

How does Primaclade work exactly?

Put simply, Primaclade takes a brute force approach to the problem of finding multiple primers. Each individual sequence in the alignment is fed into the Primer3 program multiple times to generate a comprehensive "meta-list" of possible primers for the alignment. Specifically, Primaclade runs Primer3 ten times starting with a primer length of 18 nucleotides and then increasing the length of the primer by one base pair at a time until a maximum length of 27 nucleotides. During each of these runs a maximum of 20 primers are returned creating a meta-list of 10 x 20 x (# of sequences) primers. This meta-list is then sorted, duplicates are removed, and the remaining results are checked to see if they meet the users input criteria: degenerate nucleotides and gapped-lines. If a primer falls in a region of the alignment that has the correct number of degenerate nucleotides (or less) it gets passed along. Next the primer is checked to see if the region it lies in has the correct number (or less) of sequences with gaps in it. If the correct number of "gapped" sequences occur then the primer is written into the output file.

Primaclade didn't find any primers for my alignment! What should I do now?

Increase the number of allowable degenerate nucleotides in your final primer product and try it again. Also, take a look at your alignment and pay close attention to the consensus line. In the consensus line non-degenerate sites are labeled ACGT whereas degenerate sites use the IUPAC ambiguity codes. In our experience, if sequences in your alignment are greater than 29% divergent then you aren't likely to find primers that are going to work well across it (see Tests of the Program).

I increased the Max Number of Degenerate nucleotides, but still no luck.

Try increasing the Alignment Gaps to Skip parameter. Once again, take a close look at your alignment file. One or two sequences with a number of gaps can throw Primaclade off. If you have a sequence that has several insertions that cause gap columns in the alignment, you might want to try deleting that sequence and resubmitting the new alignment. The same applies to a sequence with a large number of spaces in it. Sometimes removing just a single sequence and creating a new alignment will create a vast improvement in your consensus line.

Okay, I tried that too, but still nothing.

Last thing to try is changing the primer melting temp and the GC content parameters. Broaden the range of temps first and the GC content second. If you do all this and it still doesn't work then you might want to try splitting up your clade and looking for primers in each of the smaller alignments (see Tests of the Program).

How does Primaclade estimate the melting temperature and GC content of the primers?

The melting temp and the GC content get passed along from output that Primer3 generated for a single sequence in the alignment. Once you start including degenerate nucleotides within your primer you are going to throw those calculations off. The approximate melting temp and GC content are presented in the output merely as a rough reference point.

Where are the forward/reverse primers for my sequence?

There are no forward or reverse primers per se, there are only primers that will work across this specific alignment in this specific region. If you need to find forward or reverse primers in specific regions, run Primaclade twice and use the "Exclude Region" options. On the first run exclude everything except the right-handed area you are interested in and then run it a second time doing the same for the left hand side of the alignment.

Why doesn't Primaclade find any primers at the far end of my alignment?

Most likely because of the length of your alignment. Primaclade asks the Primer3 program to return 20 primers per sequence and it stores both the forward and reverse primers that are returned. In the normal course of events this will usually cover the entire sequence. If there are highly conserved regions at the far end of your alignment or if you suspect that there primers that should exist near the ends, try running Primaclade again and use the "Exclude Regions" function to block out the beginning of the alignment.

Does Primaclade accept IUPAC ambiguity codes?

Primaclade clade does not use IUPAC ambiguity codes when searching for primers, only when reporting the consensus sequence. Currently Primaclade takes all IUPAC ambiguity codes (and any other nucleotide information that it does not recognize) and converts it into an "N" before feeding the sequence into the Primer3 algorithm. This is actually a limitation of Primer3, and since Primaclade is built on top of Primer3 it inherits this problem as well.

Why did my results from X days ago disappear?

Primaclade result files are stored on the server for about 48 hours before the system automatically deletes them. If you are unable to access your Primaclade search files from several days ago the server has likely deleted them. Save the output file to your hard drive as soon as it is generated.

Why do all the lines in my alignment/primers output wrap around the screen?

Because your browser inserts line breaks into text that it considers to be too long. This is particularly true with Internet Explorer, Safari and Google Chrome. Firefox does not appear to have this problem. You can overcome this problem by downloading the plain text file and viewing the results in a text editor.

When I paste my alignment into the window and search for primers, Primaclade tells me "There was an error processing your alignment file."

Try uploading your file using the "Choose File" button rather than cutting and pasting the alignment.

How should I cite Primaclade?

M. D. Gadberry, S. T. Malcomber, A. N. Doust, and E. A. Kellogg. 2005. Primaclade - a flexible tool to find primers across multiple species. Bioinformatics 21: 1263-1264.

Introduction | Description | Tests | Summary | References | FAQ | Program Interface