Sequences less than 200 bp and sequences with poor overlaps (>8% difference in linked region) and/or high number of bp below a threshold score (sequences containing more than 15 bp with less than Q30, 10bp with less than Q20, or any bp with less than Q10 scores) were excluded from further analysis.
Ten clones spanned the first gap (111 bp) and 18 clones spanned the second gap (632 bp) with no less than 5× sequence coverage of any base along the length of the NotI-BamHI subclone.
Mapping results were parsed using a custom made Perl script to identify short read contigs that mapped adjacent or with a less than 21 bp identical overlap.
These genes were found to contain a 13 bp motif, with a relatively less-conserved sequence, present one or more times in their upstream regions.
Finally, a consensus sequence containing 8,948,591 bp (with an estimated error rate of less than 0.5 per 100,000 bases) that provided 25.6-fold coverage was acquired.
Another local partner will take another slice, leaving BP with less than 19%.
Only sequences longer than 550 bp with less than 1% ambiguous base calls were included (average sequence length was 692 bp), except four sequences possessing 1 3% ambiguous calls that represented the sole records for their species.
A total of 4000 sequences were analyzed, only sequences of more than 170 bp with less than 5% ambiguity were processed.
However previous studies were based on only partial (~500 bp) 16S rRNA gene sequence analysis, whereas for taxonomical purposes the complete gene should be sequenced (>1300 bp with less than 0.5% undetermined bases) [ 5, 6].
Full length 16S rRNA gene sequences, comprising greater than 1300 bp with less than 0.5% undetermined positions, were obtained for 66 E. sakazakii strains representative of the different biogroups and 13 strains representative of other species.
