It has been shown that hybridization conditions affect the specificity for single nucleotide mismatch, such as hybridization temperature [ 34], time [ 35, 36], washing stringency [ 37, 38], and dimethylsulfoxide, formamide, etc., included in hybridization solution [ 39].
These processed PnFL2 sequences were assembled with the phrap program (Laboratory of Phil Green, Genome Sciences Department, University of Washington) [ 32] under default conditions, except for the following criteria: -minmatch 400, -minscore 400, -repeat stringency.999, -trim quality 20.
Single isolated colonies from each plate were picked, rigorously vortexed in 500 µl low stringency medium, from which 5 µl drops were placed onto low (SD/−2), medium (SD/−3) or high (SD/−4) stringency selection plates and incubated 3 days at 30°C.
The "build" option was used to order markers within the linkage group, with the same 1000 1 stringency as for the RH map (PUK_LIKE_TOL set to 3.0).
Base calling from traces and quality scores assigned using Phred [ 60, 61], and Phrap used to assemble the sequences into contigs (200 minscore, 0.99 stringency; http://www.phrap.org/).
Genes were clustered into 'tribes', an approximation of gene families, using TRIBE MCL [ 63, 93, 94] at medium (Inflation I = 3.0) stringency.
Contigs larger than 200 bp from both datasets were assembled using Phrap (200 minscore, 0.96 stringency), with resulting contigs 400 bp or larger retained.
After 2 stringency washes (0.5 × SSC with 0.1 % SDS buffer at 65 °C for 15 min), detection was carried out using a block buffer and the anti-digoxigenin-alkaline phosphatase conjugate antibody (Roche, Germany), following the manufacture's protocol.
For example, for V$NOLF at the "opt-0.1" stringency, we find 653 matches in the 200 bp preceding the TSSs of the 314 non-redundant ORs, but only 198 matches in the preceding 200 bp, representing ~3.3-fold enrichment.
At ≥−1.0 stringency level, the fuzzy nucleosomal regions are 8-10 folds higher than the distinct nucleosomes in terms of the number of nucleosomes (and 36-39 times higher in terms of number of bases covered).
Thus, our results are in good agreement with an already published nucleosome map of Adh gene [ 48], thereby justifying the ≥−1.0 stringency criteria experimentally, to assign nucleosome positioning genome wide.
