with ProAssembler default settings (match size = 25, minimum match percentage = 80, match spacing 150, minimum sequence length = 100, gap penalty = 0, gap length penalty = 0.70, and maximum mismatch end bases = 15).
Requiring either multiple independent k-mer matches from a single family or a minimum number of k-mer matches over a minimum sequence length is used to adjust the sensitivity of the match.
These datasets were then assembled using the SeqMan Pro assembler of the Lasergene software package v8.0.2 (DNASTAR, Madison USA) with the following program parameters: match size, 50bp; minimum match percentage, 80%; minimum sequence length, 40 bp; gap length penalty, 0.70 and maximum mismatch end bases, 15.
Assembly for contigs was performed with match size of 50, minimum match percentage of 97, minimum sequence length of 100.
The options employed for SeqMan II assembly were match size = 12 bases, minimum match % = 80, minimum sequence length = 100, maximum added gaps per kb in contig = 70, maximum added gaps per kb in sequence = 70, maximum register shift difference (maximum base pair separation) between matches = 70, gap penalty = 0, and gap length penalty = 0.7.
Following assembly, chloroplast contigs were merged using the minimus2 application of the AMOS 3.1.0 assembly package [ 26] running default parameters and for the titration of depths of sequence coverage, SeqMan (LazerGene) using a match size of 5, a minimum match percentage of 95 and a minimum sequence length of 1000.
The remaining 1037 sequences were assembled into clusters (≥two ESTs) and singletons (one EST) based on the following criteria: sequence match size of 20 bp, minimum match percentage of 85%, 0.00 gap opening penalty, 0.7 gap length penalty, and minimum sequence length of 70 bp.
At a minimum of 50 out of 69 bp (72%) sequence match, 52 of the 59 outlier markers returned BLAST matches to the 11 main scaffolds of the E. grandis genome.
The EST sequences were assembled into contigs using CAP3 [ 9] under high stringency parameters of a minimum sequence identity of 98%; minimum overlap length of 40 nt; gap penalty, 6; match value, 2; mismatch penalty (−5).
A rapid, high stringency clustering was performed first, using the "Fast Assembler" module, with the following parameters: minimum match 90%, match size 25, match spacing 150, gap penalty 0, gap length penalty 0.7, end position mismatch 0, and minimum sequence length 50.
