The map was built with a relatively relaxed alignment cut-off value of 1e-21.
The final map was built with a more relaxed alignment cut-off value of 1e-09, followed by removal of entanglements with the DQ-er function at cut-offs of 1e-10 to 1e-12.
The BAC contigs were built with a slightly relaxed alignment cut-off value of 1e-09, which maximised the number of incorporated BACs, but also resulted in a high initial number (11221) of questionable fingerprints, with friction alignment.
For the set of restriction sites from step 1, or as provided by the user, all valid upstream, downstream and, if desired, hybridization probe primers within a scan window are enumerated, considering the user-defined parameters for GC-content, melting temperature and self alignment cut-offs.
On the other hand, setting of high alignment cut-off thresholds (score = 40) resulted in the efficient highlighting of only those coding regions with a significant similarity to sequences in databases, a pool of genes that are easily identified by conventional methods.
An optimal physical map quality, with relatively large contigs containing few questionable clones was obtained with a band size tolerance of 5 (which corresponds to a 0.5 bp size difference in the fingerprints) and an alignment cut-off value of 1e-11.
The alignment was cut to the region of interest (amino acid 180 220) and gaps were removed.
The alignment was cut from E. coli position 430 (totally conserved GTAAA) and reference sequences, except for Methanobrevibacter smithii, were then deleted from the alignment with BioEdit version 7.0.5.3 [ 30].
Amino acids were searched against the UniProtKB/Swiss-Prot database using an E value cut-off = 1e-6 (retaining best 20 hits), with a minimum amino acid alignment length cut-off (high-scoring segment pair length) of 33.
Reviewer 1 The input alignment used cuts out both N- and C-termini of the proteins which may have appeared messy in the large alignment due to strongly differing terminal extension lengths.
