Ortho-SSR-Euca

News SSRs Pipeline Target

• Designing Multiplex SSR primers from E. camaldulensis, E. grandis and E. globulus available EucalyptusDB and NCBI sources
• Author: Nagabhushana and Rajkumar
• Timeline
  • Designing Primers: DEC 2009
  • Oligo synthesis  : JAN 2010
  • Validation  : FEB 2010
  • Manuscript  : MAR 2010

Scientific Assumptions

• Given the situation where E. camal DNA sequences are not publicly available, exploit the E. globulus (ESTs) and E. grandis (scaffolds) by mapping the ESTs on scaffolds, findout the conserved region in scaffolds and develop markers. These markers are expected to work well in E. camal due to the genome synteny across species

Strategy

• Globulus ESTs
• Find Repeats and filter the ESTs
• Assemble ESTs with repeats as Uni genes
• Map the repeats based globulus unigenes on E. grandis scaffolds
• Again find the repeats on those scaffolds
• Mark the +/- 300 bp coordinates of the repeats
• Design multiplex primers (to save 3 folds of time and money) marking the repeat region
• Validate the primers on E. camal experimentally.

Implementation

• EST from E.globulus: total 13468

Trimming

• Removed the seq with < 50 bp
• Removed the seq that have more than 80% of N
• Replaced '-' or 'N'
• Resulted Sequences : 13441
• Trim_fasta.pl

Repeat Prediction

• Param
  • definition(unit_size,min_repeats): 2-6 3-5 4-5
• Implementation MISA
• Results
  • Total number of sequences examined: 13441
  • Total size of examined sequences (bp): 8544644
  • Total number of identified SSRs: 2330
  • Number of SSR containing sequences: 1873
  • Number of sequences containing more than 1 SSR: 387
  • Number of SSRs present in compound formation: 290
  • Repeats: Di -> 1159; Tri -> 1128; Tetra -> 43
• scripts
• misa_run.pl (Few sec)
• parse_misa.pl (15min)

TGICL Contig assembly

• Input: SSR glo ests:1873
• Param
  • 4 Core threaded (implementation was multithreaded)
  • minimum percent identity 90
  • miminum overlap length 35
  • maximum length of unmatched overhangs 30
  • Cap3 based All-vs-all search
  • Contigs were filtered off for ESTs with more than 4
• Results
  • globulus_repeat_unigenes: 301
  • globulus_repeat_contigs : 130
  • globulus_repeat_singlets: 171
• Time Taken
  • 1 mins: 18 secs under 4 core (of 2.8GHz AMD Phenom HT) threading
• Scripts Written
  • tgcl_run.pl => multithread implementatin of TGICL
  • tgicl_parse.pl => joins the results as unigenes contigs and singlets from each thread

Blast

• blastn | query: 301 Globulus Unigenes | target: EucaDB scaffolds
• GUI based blastn | Output is saved as text and parsed
• Parsing
  • Parse the Blast output of scaffold mapping
  • Write the results into a seperate folder with each EST
  • Write the statistics files (est_scaf_hit.stat, unique_scaf_hit.stat)
  • Script : parse_blast.pl | Took 5 sec in Quadcore 2.8GHz machine
• Results
  • Total number of ESTs with hits: 301
  • Total number of unique scaffolds: 173

Get the co-ordinates of Grandis scaffolds

• Downloaded the 173 scaffolds
  • Made the clear fasta file as ALLCAPs sequences caps_fasta.pl
• Parse the Blast out to get the co-ordinates
  • Parse the parsed Blast output of scaffold mapping
  • Get the hit co-ordinates for each scaffold
  • The co-ordinate window of EST-scaffolds map that have only 3kb in length
  • Write the results as scaffold and co-ordinate window
  • Pick out the selected scaffold sequences only with that co-ordinate window
  • Some scaffolds have distinct hits at more than 1 places and will written as separate file. They need to cured manyually.
  • co-ordinates_blast.pl | Takes 2 sec
• Results
  • Number of scaffolds hit: 284
  • Number of unique scaffolds: 160
  • Number of final scaffolds with single region hit : 134
  • Number of multiple scaffolds with single region hit: 14
  • Total number of workable scaffolds to deal for SSR : 166

Mark and Fetch the Scaffolds with desired co-ordinates

• Select the scaffolds
• Mark the specific region
• Add 300bp on both the sides
• Fetch the scaffolds as fasta
• Script fetch_scaf.pl

Make the input file for final SSR Pipeline

• Make the available camal DNA sequences, Trim with more than 600 bases
• Combine the final Grandis and Camal sequences together
• Result: Total : 461 of which Gran: 148 and Camal: 313
• Script: make_data_file_ssr.pl

Gene Prediction using Genscan

• Genscan Run on Final output of 461 sequences
• Results: All fasta are known to contain exons

Repeat Prediction

• Param
  • print OUT "PRIMER_MIN_GC = 49\n";
  • print OUT "PRIMER_MAX_GC = 55\n";
  • print OUT "PRIMER_OPT_GC = 50\n";
  • print OUT "PRIMER_MIN_TM = 59\n";
  • print OUT "PRIMER_MAX_TM = 63\n";
  • print OUT "PRIMER_OPT_TM = 61\n";
  • print OUT "PRIMER_OPT_SIZE = 22\n";
  • print OUT "PRIMER_MIN_SIZE = 21\n";
  • print OUT "PRIMER_MAX_SIZE = 23\n";
  • print OUT "PRIMER_NUM_RETURN = 1\n";
  • print OUT "PRIMER_PRODUCT_SIZE_RANGE=90-320\n";
  • definition(unit_size,min_repeats): 2-9 3-6
  • interruptions(max_difference_between_2_SSRs): 100
• Implementation MISA
• Results
  • Total number of sequences examined: 461
  • Total size of examined sequences (bp): 700099
  • Total number of identified SSRs: 169
  • Number of SSR containing sequences: 124
  • Number of sequences containing more than 1 SSR: 36
  • Number of SSRs present in compound formation: 14
  • Repeats: Di -> 120; Tri -> 48
• scripts
• misa_pipe.pl (~20 sec)

Primer Design

• Single Plex
  • Issue: Designing with the broader range 100-350 thows more into higher side
  • Strategy: 4 ranges indenpendent pipeline : 100-150 | 160 210 | 220-270 | 280-330
    • Pick up 4 equal ranges from camal and grandis
    • Ignore primer pairs based on high difference in For/Rev Tm value
    • Type of Repeats in the primer
    • ePCR positive ness
• Multiplex
  • PrimerPlex Dependency