Locating sequence on FPC maps and selecting a minimal tiling path

Abstract

This study discusses three software tools, the first two aid in integrating sequence with an FPC physical map and the third automatically selects a minimal tiling path given genomic draft sequence and BAC end sequences. The first tool, FSD (FPC Simulated Digest), takes a sequenced clone and adds it back to the map based on a fingerprint generated by an in silico digest of the clone. This allows verification of sequenced clone positions and the integration of sequenced clones that were not originally part of the FPC map. The second tool, BSS (Blast Some Sequence), takes a query sequence and positions it on the map based on sequence associated with the clones in the map. BSS has multiple uses as follows: (1) When the query is a file of marker sequences, they can be added as electronic markers. (2) When the query is draft sequence, the results of BSS can be used to close gaps in a sequenced clone or the physical map. (3) When the query is a sequenced clone and the target is BAC end sequences, one may select the next clone for sequencing using both sequence comparison results and map location. (4) When the query is whole-genome draft sequence and the target is BAC end sequences, the results can be used to select many clones for a minimal tiling path at once. The third tool, pickMTP, automates the majority of this last usage of BSS. Results are presented using the rice FPC map, BAC end sequences, and whole-genome shotgun from Syngenta.

Figure 1

SD clones and electronic markers. The clones in blue are simulated digest clones from the Japanese minimal tiling path; most of the original clones are not in FPC. The markers in blue are electronic markers supplied by Gramene. As described in the text, these markers could have been added by BSS. The contig display is from FPC V7. Tracks can be added, resized, and moved around.

Figure 2

The windows that would be shown for a Marker→Sequence search using MegaBLAST in the Batch mode, which runs the search on all contigs. Values are entered in the Setup and Batch BSS windows and the search started. The results can be viewed in the BSS Report window. If the results are to be automatically added as markers using the Add as FPC Markers function, the Marker Add Conditions may first be altered.

Figure 3

Selecting the next clone for sequencing. Clone AP005522 has been sequenced. It was blasted against all of the BESs in the contig. The clone was added as a marker attached to all of the clones that had a BES hit. Selecting the AP005522 marker highlights these clones. Some clones are obviously contained in the sequenced clone, so can be ignored. The BSS report file can be viewed to see details of the hits.

Figure 4

The distribution of expressway lengths.

Figure 5

The five different types of pairs. Case 1 pairs consist of two overlapping clones, with the exact amount of overlap identified by the seqCtg. Case 2 clones have a gap between them; they are bridged by a seqCtg. In Case 3, the two clones cover a similar area. Case 4 pairs result from false positive hits. Case 5 pairs result from two small clones hitting on opposite ends of a long seqCtg; note this is similar to Case 1, but a large seqCtg may result in a large overlap.

Figure 6

(A) S1 and S2 are sequenced contigs, and C1, C2, and C3 are clones. The underlined sequence represents the BESs and where they hit on the seqCtg. Note, all lengths of sequences are tremendously reduced to fit on the page. (B) There are four possible pairs, shown in the four rows in the table. A + indicates the BES was not reverse complemented to match the seqCtg. An rc indicates that it was reverse complemented. The seqCtg-BES hits for the first two pairs both have the same orientations, whereas the orientations for the next two are different. The first two are rejected as candidate MTP pairs, wheras the second two are retained.

Figure 7

Construction of DAGs from overlapping clone pairs. (A) The amount of overlap or distance is recorded for all good clone pairs, shown as w, x, y, and z. (B) Clones define vertices, and pairs define edges, with edge weight determined by overlap or distance. If there is a gap between the clones, then the value is multiplied by 10. An MTP, shown in gray, is picked from the expressways. Note the junction from C to F, in which there is no sequence evidence that the clones overlap.