The spreadsheet file contains new or revised insertion site data insertion site data and flanking sequences for 42 more Exelixis lines. There are BDSC stocks for 38 of these lines. The common thread for these lines is that I either submitted new flanking sequences to GenBank or revised the existing GenBank sequence. I have expanded my standard spreadsheet with respect to the flanking sequences; I put in separate columns for the 5' and 3' flanks for pre-existing, new, and updated GenBank accessions. I tried to keep the rest of the spreadsheet the same as for the last batch of Exelixis lines. The flanking sequences are consistent with two sites on chromosome arm 3L for c04241. I entered c04241 and c04241-3 as the line ID's for these two rows in the spreadsheet. The row for lineID c04241 corresponds to the site for the existing FlyBase record (FBti0041133) and the site reported by Exelixis in CG5910. The new 5' flank maps to this site. The c04241-3 site is based on the previously submitted 3' flank. There are at least five other Exelixis insertions at this same site in FlyBase, so either this is a hotspot, cross-contamination, or some kind of an artifact. The d08138 line was reported by Exelixis to map to a unique site in the HPS4 gene. The GDP database for this P-element line has four 5' flank sequencing runs (imported from Exelixis) and four 3' flank runs. The best quality of the 5' flanks is 149 bp ending in the restriction site used for iPCR. The other three 5' flank runs are identical to this for varying distances, depending on the sequencing quality. The 5' flank aligns 149/149 bp to many sites in the reference genomic sequence and to the consensus for TE 1360. The four 3' flanks are identical 7 bp sequences ending in the restriction site used for iPCR. The 5' and 3' flanks share the same target site duplication, consistent with them originating from the same insertion site. My conclusion is that the Exelixis mapping to a unique site is incorrect and that this P-element is inserted in a copy of TE 1360 at an unknown site. The other 40 insertion lines in this batch have a single insertion site, as far as we can tell from the flanks, and the site is close to that reported by Exelixis. I am working on three other lines in which the flank maps to a different chromosome than reported by Exelixis and I plan to resolve those conflicts by genetic segregation tests. The flank for f07325 aligns to two sites in a local duplication and the two sites are in different genes. Exelixis had published these two possible sites. There are 12 lines in this batch for which we cannot read or align the flank(s) all the way to the vector-flank junction. Consequently, we have not determined the precise sequence location of the insertion site. I have indicated this in the Genome Position Comment and have entered a minimum, maximum, or range of locations. I discovered that the 3' flank for the e00405 line that was previously submitted to GenBank (accession CZ478292) aligns entirely with the vector and may indicate a vector-internal rearrangement involving the 3' end. The new 5' flank that I submitted does not extend to the vector junction, but aligns near the site reported by Exelixis. The entries in the GDP Upstream Gene column are insertions upstream of the annotated transcription unit, but within 500 bp of the 5' end.
