Explored different programs that could design multiplex PCR primers for our sequencing project of transcriptome of Pteronotus parnellii. Have collection of intronic regions from genome. Trying to design primers to make reads of 100-200 bp. This will be a single template PCR reaction, as all reads come from the same organism, jut different parts of the genome.

Important things to remember in multiplex PCR primer design (in addition to general PCR primer design) via PremierBiosoft:

• Primer length should be shorter (18-22bp). Do not want to go shorter than 15bp.
• Melting temperature between 55-60 degree C.
• Highly specific (more important for multiple target multiplex).
• Avoid primer-dimers. 

Nothing seems too new.

After searching for programs, only few claimed to be able to hand multiplex primer design. Nearly all use the Primer3 algorithm. Nearly all programs were not open source. Here are the attempts:

• PerlPrimer: looks promising but I did not have a Unix machine on hand. Download with GUI for Windows would not work at all for mysterious reason. Tried running on Cygwin but many of the tests were failing so I figured I would keep checking.
• MPprimer: also looked very promising but only works on Unix machine
• jPCR: java app that actually works great, but think it works best for smaller sequences or more similar sequences. I didn't try running on the server so that could help, but I could only get primer designs for up to ~30 sequences before freezing.
• Geneious Pro: (sigh) end up using the fancy-schmancy. Although doesn't have multiplex primer option (allegedly coming soon), was able to load 900 sequences as a sequence list and design primers for about 450 sequences and over 4,000 primers. Making target region smaller, primer length shorter, and narrowing GC content helped narrow down, but still need to sift through some of the sequences.

To-Do List:

• Write script to sift out intron sequences that are too repetitive (or is there one out there already?)