Therefore, a minimum target sequence size (long SSRs or high density of short loci) is required for ND-FISH signals to be detected even when using polytene chromosomes.
BLAT with a tile size of 7 and minimum sequence identity of 80% was used to find read hits to the remaining putatively nuclear masked unigenes.
The mouse genome sequence had already been soft-masked for repeats by UCSC and BLAT was set to produce all possible alignments (tile size = 10, minimum score = 0, minimum sequence identity = 0).
Assembly for contigs was performed with match size of 50, minimum match percentage of 97, minimum sequence length of 100.
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).
Scaffolds having a sequence length of 2 kbp or more covered about 96%% in size of the de novo genome assembly sequence of which 17 scaffolds had a minimum sequence length of 0.5 Mbp (Additional file 5: Figure S1).
Minimum sequence similarity threshold was specified in terms of percent identity (-S parameter) and varied between 10 and 80 with step size 10.
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.
Default parameters were used for BLASTN searches, with the following exceptions to account for the divergence and short length of the sequences available: minimum alignment size 80 nt, minimum percentage of sequence identity 25%%, maximum e-value 0.001 and low complexity mask on.
