DOE Human Genome Program Contractor-Grantee Workshop VIII
February 27-March 2, 2000  Santa Fe, NM

110. Contamination of BAC Clones by E. coli IS186 Insertion Elements

Owatha L. Tatum, Andrew W. Womack, Mark O. Mundt, and Norman A. Doggett

Bioscience Division and DOE Joint Genome Institute, Los Alamos National Laboratory, Los Alamos, NM 87545

doggett@lanl.gov

The E. coli insertion element IS186 is a 1343 bp transposable element which is present at three to four copies in the E. coli genome. The transposon is flanked by a 23 bp inverted repeat and has been shown to insert preferentially into GC-rich targets. We have discovered 8 BAC and P1 clones from several widely used human genomic libraries which have a single copy of this insertion element included in the finished Genbank submission. These clones were sequenced at six different sequencing centers and in no case was the insertion element annotated as being derived from E. coli. The average G+C content of a 100 bp window on either side of the insertion site in all clones is very high (75.8%) and appears to be within CpG islands. In two of the 8 cases the insertion site is flanked by G+C-rich SVA repeat elements (a retroviral LTR class of repeat). Earlier studies of IS186 insertions into plasmids have shown that target duplications of 8 to 12 bp occur at the insertion site. We looked for evidence of target duplication in a BAC clone containing this insertion by comparison with the finished sequence of a cosmid clone which overlapped the insertion site. This proved that the insertion of IS186 caused a 10 base pair duplication of human sequence surrounded the insertion site. Ten base pair duplications were surrounding the insertion site were found in the finished sequence of all other clones. In order to determine whether IS186 insertions occurred during library construction or propagation we performed PCR and sequencing experiments on several isolates of each clone. We found that RPCI-11 BAC clones sent directly from the Roswell Park resource did not contain insertion elements providing strong evidence that IS186 insertions are most likely occur during subsequent propagation of BACs. We estimate the frequency of this insertion in finished clones to be approximately 1 in 1000 but the actual frequency could be much higher if this element has been removed from some finished sequences prior to submission.

Supported by the US DOE.