Casuarina SSR

New SSRs Pipeline Target

• Designing Cross species EST SSRs from Casuarina
• Author: Ashok Kulkarni and Rajkumar
• Timeline
  • Designing Primers: DEC 2011
  • Oligo synthesis  : JAN 2012
  • Standardisation and Validation  : MAR 2010
  • Manuscript  : JUN 2010

Scientific Assumptions

EST SSRs are supposedly conserved across species therefore higher species transferability is expected

Strategy

• C. glauca ESTs
• Develop unigenes
• Generate statistics
• Filter the sequence
• Mine for repeat motifs
• Design Primers

Implementation

C. glauca ESTs

• Total Number of ESTs: 34897
• Total number of C. glauca ESTs: 34805

Develop unigenes

• TGICL is used
  • Param: Left to default

# -l miminum overlap length (default 40)
# -M ignore lower-case masking in <fasta_db> sequences
# -p minimum percent identity for overlaps <PID> (default 94)

• Contigs : 3947
• Singlets: 475
• Unigenes: 4422

Generate statistics

• Final number of sequence
  • Param: Trimmed off the <220bp | Trimmed off the N>20%
  • ID Named
• Distribution of length in contigs: 220-500: 546 | 500-1000: 3119 | 1000-1500: 529 | >1500: 98
• Trimmed Sequences: 4285

Mine for repeat motifs

• Param:

Definement of microsatellites (unit size / minimum number of repeats): (2/7) (3/5) (4/4) 

• Stat

Total number of sequences examined:              4285
Total size of examined sequences (bp):           3253599
Total number of identified SSRs:                 383
Number of SSR containing sequences:              347
Number of sequences containing more than 1 SSR:  30
Number of SSRs present in compound formation:    24

Distribution to different repeat type classes
---------------------------------------------

Unit size	Number of SSRs
Di	217
Tri	141
Tetra	25