(For those of you who may have forgotten, Solexa sequencing is a rapid, highly automated method of generating millions of short sequences at random across a DNA sample — often, an entire genome).
I have just received the first set of Solexa data from our collaboration with Fritz Roth and his colleagues, Yong Lu & Joe Mellor. The image below shows “read depth” (the number of runs which cross a given point) in the neighborhood of lacZ for strains TT24815 and TT25790. We expect this measurement to increase in proportion to the degree of amplification. Coverage over non-amplified areas of the plasmid and chromosome exceeded 50-fold for both strains.
Small red arrows show my guess at the amplification endpoints. The TT24815 array stretches from approximately 138256 to 166250 (~28 KB), and TT25790, from 131600 to 159300 (~28 KB), where the coordinates refer to our standard F’128 sequence counting clockwise from the first nucleotide of IS3A.
Strain TT25790 contains Elisabeth’s known inversion duplication array (EK568), for which we have sequenced a single join point (134075->134087 recombined into 132108<-132098). Small blue arrows show these two tracts. In our simple models of inverted duplication formation, join 1 forms from their recombination, either directly (“Flying Walendas”) or by assymetric deletions of a larger toxic structure (“Slytherin”). Furthermore, all Solexa data in the array should begin at the leftmost blue arrow, gratifyingly close to my guessed endpoint. Join point 2 will be defined by a sequence near the righthand red arrow and its inverted complement at a position yet to be found in the amplified region. I shall go hunting!
Strain TT24815 was chosen for its recalcitrant nature — we were never able to find any join points, but assumed for this reason that it was a likely candidate for an amplified inverted duplication, as crossover sites in these entities are truly difficult to locate and sequence. We were hoping to get two new bits of previously unknown information out of it. Once again, half of each of the join points should be defined by small sequence inversions in the neighborhood of the red arrows, assuming that this is a truly simple array of elements representing one kind of inverted duplication. More hunting!
If you use your browser’s zoom feature, you can inspect the image with better resolution. You can also download a detailed PDF file.
-- Eric Kofoid