OK, so I got to my office this morning all enthusiastic to do the additional runs that would clarify why N. meningitidis has twice as many forward-orientation DUSs in the strand synthesized discontinuously. I did three different things, all of which confirmed that the two-fold difference was just an aberration in the Gibbs analysis.
First, I plotted the distribution of forward-DUSs along both strands of the genome (yesterday I only had time to do it for the reverse-complement strand). This clearly showed that the two strands are the same-- the blue ticks in the figure below (just a close-up of part of the figure) are DUSs on the forward strand, and the red ones are DUSs on the reverse-complement strand.
Second, I completed the control analysis I had to interupt yesterday. This analyzed the reverse-complements of the 'leading' and 'lagging' sequences I had assembled yesterday. It was a way of repeating the analysis on different sequences that had the same information content. Result: very similar numbers of DUSs in both.
Third, I assembled new 'leading' and 'lagging' sequences, using our SplitSequence.pl script to efficiently find the midpoints I'm using as surrogate termini, then reran the Gibbs analysis on these. Result: very similar numbers of DUSs in both, and these DUSs gave effectively identical logos.
So I went back and examined the Gibbs output that had had twice as many DUSs as the others. For unknown reasons, both replicate runs had settled on less-highly specified motifs, and thus included a lot more poorly matched sites in their output. Well, at least I can now very confidently report that there is no direction-of-replication bias in N. meningitidis DUSs.
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in The Biology Files
Not your typical science blog, but an 'open science' research blog. Watch me fumbling my way towards understanding how and why bacteria take up DNA, and getting distracted by other cool questions.
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