Clone-based strategies to sequence highly repetitive regions. (a) Novel transposon-based strategy [see also Szybalski et al. (36)]. (1) In vitro transposition of Tn599 into BACR26J21 generates a library of clones that carry this transposon at random positions. A and B represent clones where Tn599 is inserted at different positions. (2) PI-SceI digestion of each clone generates two DNA fragments. (3) In the case of A and B, the two sets of fragments are identical in size, as can be seen in the PFGE image. (4) In order to distinguish between the two putative positions of the transposon in the clone, a Southern blot analysis is performed, using the probe P, that marks the fragment that carries most of the vector used in the construction of the BAC library. (5) Sequencing of each clone is performed using two primers reading outwards from both ends of the transposon. The reads are then assembled using both positional and sequence data. (b) Current strategy. (1) Collect shotgun sequence data and assemble the data into sequence contigs using computer programs. (2) Assess the likelihood of resolution before committing to the LIL-TIL strategy. (3) Generation of a Large Insert Library. Large insert subclones are then chosen for TIL based on overlap in sequence believed to occur only once in the clone. The diagram illustrates how, by choosing a number of subclones that overlap in unique sequence, a minimum tile path can be built to correctly assemble large sections of repeats. (4) Build each LIL subclone using a Transposon Insert Library (TIL). (5) Combination of the LIL-TIL strategy, existing read-pair information and restriction digest data to build a correct assembly.

Distribution of 620 insertions of Tn599 along the BACR26J21 is represented on the horizontal axis of the graph. Positions of the insertion sites within the BAC clone are shown in the vertical axis. The black line represents perfect randomness in the distribution of the insertions.

Sequence analyses of BACR26J21. (a) Self comparison of the BACR26J21 sequence shows the presence of tandem repeat sequences. Circles indicate the positions of tandem repeated sequences (b) Dot plot analysis of the first 95 kb of BACR26J21 (x-axis) against the 3.1-kb sequence of the repeat unit of the 18 HT satellite (y-axis) generated using Dotter (21). Asterisks indicate the positions of deleted units. The positions of the transposons 1360, 412 and copia are also indicated. Numbering is in base pairs.

Illustration of the Y centromeric region h18 encompassing the 18HT satellite. (a) Prometaphase Y chromosome counterstained with 4′, 6-diamidino-2-phenylindole (DAPI). The centromere is indicated with a C and the regions h17 and h18 are also indicated. (b) Schematic representation of region h18 showing the 18HT satellite and the extent of the assembled contig. The position of the gene Pp1Y2, the pseudogenes Mst77F and some retrotransposons are also indicated. The names of the BACs are indicated. BACR26J21 appears at the bottom. (c) Structural organization of the 18 HT satellite repeat unit. The 3.1-kb tandem repeat unit contains three truncated 3′ UTRs of HeT-A and one truncated 3′ UTR of TART.

Mapping of the Tn599 insertions. (a) PFGE image of a set of clones (lanes 2–16) carrying the transposon, after digestion with PI-SceI. (b) Corresponding Southern blot hybridized with labeled probe. Data from (a) and (b) together allows the correct mapping of the transposon in each clone. Lanes 1 and 17 corresponds to Low Range PFGE Marker (NEB) and Lane 18 is Mid Range I PFGE Marker (NEB). Run conditions were 6 V/cm, 14°C for 20 h and a pulse time ramp of 0.4–10 s.

Comparative analysis of the two sequence assemblies of BACR26J21. (a) Diagram showing the location of the nucleotide differences between the assemblies (CU076040 versus FM992409). The assembly that utilizes positional information appears at the bottom (FM992409). In order to facilitate the comparison, different colors are given to different transposons. (b) Sequence showing that the extra 33 nucleotides (in red) are located in the middle of a 31-bp palindrome (in black bold).

Parsimonious reconstruction of the evolution of the centromeric region of the Y chromosome of D. melanogaster. (a) Schematic representation of the sequence of the centromeric region h18 of the Y chromosome, not to scale. The sequence of BACR26J21 was analyzed by BLAST through FlyBase and by dot plot analyses. Slim orange arrows represent HeT-As decayed by deletions and/or few small inversions. A yellow arrow (or fragment of it) represents 3′ UTR TART sequences. Pentagons (rightward open arrowheads) including HeT-A and TART sequences represent units of the 18HT satellite (Figure 1c). Each colored triangle represents a different transposon, with its orientation indicated by a thin black arrow, and its name displayed on top. All transposable elements are degenerate in comparison to the canonical element (http://flybase.org/), except 1731, which has been recently inserted. Green squares represent a small segmental duplication from the euchromatic region 42A. (e) to (a) is the order of the inferred evolutionary steps. Amplifications and deletions of various regions are indicated by two different types of discontinuous lines. For example, the region including sequences of four telomeric elements went through amplification, giving rise to the 18HT satellite, (e) to (d). Insertions of transposable elements are shown with triangles and the insertion of the sequence from 42A is indicated by a blue dotted line, (d) to (c). The outcome of three deletion events within the units of the 18HT satellite is indicated by contraction of the pentagon, (c) to (a). Violet parentheses have been used to reduce the diagram of the 18HT satellite region. (e) Schematic representation of the ancestral telomeric region. The telomeric array was composed of nine different telomeric elements, numbered from 1 to 9: four truncated HeT-As (1, 2, 3 and 5), one truncated TART (4) and four full-length HeT-As (6, 7, 8 and 9). Numbers have been preserved all along the scheme in order to help visualize the amplified regions.