C. Briggsae Genotyping Primers
We successfully designed FP-TDI genotyping assays for ~19,000 of the 23,000+ SNPs in HK104 "build 4".
Repeat-masked, SNP-marked sequence was used to avoid polymorphic/repetitive primer sites.
Assays include chromosome and ultracontig coordinates for the relevant SNP
from the "cb3" and "cb25" sequence assemblies, respectively. The older assays (designed in 2005) are at the bottom of this page.
Genotyping with FP-TDI
Genotyping with the FP-TDI platform requires at least three oligonucleotide primers. Two PCR primers (p1 & p2) are used to amplify a region containing a SNP of interest.
Next, an extension primer hybridizes to the sequence immediately proximal to the SNP locus. Extension primers can be designed upstream (p3) or downstream (p4)
of the SNP.
Repeat Masking and PCR Primer Design
We used open source RepeatMasker software to mask repetetive elements prior to PCR primer design. Our C. briggsae organism-specific repeat library is available upon
request. We used the primer3 package (another freely available application) to select PCR primers at a standard set of protocols.
SNP (TDI) Primer Design
The extension reaction requires a single TDI primer that anneals immediately proximal to the SNP (called 'p3' for forward orientation, 'p4' for reverse
orientation). TDI primers are designed using unmasked flanking sequence, since a PCR product of 80-400 bp contains the target for TDI primer hybridization.
We design TDI primers to be 15-40 mer oligos with a TM between 50 and 55.
TDI primer design is always attempted for both orientations; only the orientation that was used for genotyping is provided.