Summary
A total of 13175 C. canephora unigenes were downloaded from the internet
database( ftp://ftp.sgn.cornell.edu/coffee/). The sequences were
screened for the presence of di, tri tetra, penta and hexa nucleotide
repeat motifs using MISA perl scripts
(http://pgrc.ipk-gatersleben.de/misa/download/misa.pl).
The parameters are configured to have minimum threshold of 2-8 3-6 4-5 5-5 6-5
(di repeats 8 times). The SSR co-ordinates were marked on the unigenes and
primers were designed on the flanking region so as to have PCR product
length of ___ to ___ using Primer3
(http://sourceforge.net/projects/primer3) perl scripts with default
parameters. The complete perl pipeline involves series of steps viz.,
idenfying the repeats, marking the repeat co-ordinates, parsing the
results, converting the results into Primer3 input format, Running
Primer3, parsing and converting the Primer3 output in GFF format. The
entire pipeline was automated using perl, while each step was considered
as a module.
Of the 13175 sequences examined 510 SSR's were identified in 476
sequences. 31 sequences conrtained more than 1 SSR. The primers were
further filtered used desired primer model. The results obtained are
summarised in the Table below
Analysis
- The Coffee EST data can be accessed here
- The results can be accessed here