Abstract
BackgroundSleeping Beauty transposon (SB) has become an increasingly important genetic tool for generating mutations in vertebrate cells. It is widely thought that SB exclusively integrates into TA dinucleotides. However, this strict TA-preference has not been rigorously tested in large numbers of insertion sites that now can be detected with next generation sequencing. Li et al. found 71 SB insertions in non-TA dinucleotides in 2013, suggesting that TA dinucleotides are not the only sites of SB integration, yet further studies on this topic have not been carried out.ResultsIn this study, we re-analyzed 600 million pairs of Illumina sequence reads from a high-throughput SB mutagenesis screen and identified 28 thousand SB insertions in non-TA sites. We recovered some of these non-TA sites using PCR and confirmed that at least a subset of the insertions at non-TA sites are real integrations. The consensus sequence of these non-TA sites shows an asymmetric pattern distinct from the symmetric pattern of the canonical TA sites. Perfect similarity between the downstream flanking sequence and SB transposon ends indicates there may be interaction between the transposon DNA binding domain of transposase and the target DNA.ConclusionThe TA-preference of SB transposon is not as strict as what people had thought. And the SB integrations at non-TA sites might be guided by the interaction between the transposon DNA binding domain of SB transposase and the target DNA.
Highlights
Sleeping Beauty transposon (SB) has become an increasingly important genetic tool for generating mutations in vertebrate cells
In 2005, Yant et al identified more than 1300 SB insertions, which all targeted to TA dinucleotides [11], further confirming the strict TA
Twenty-eight thousand integrations at non-TA sites were identified by re-analyzing previous sequencing data To search for possible integrations in all genomic contexts besides TA sites, we re-trimmed the sequence reads from a previous study that sequenced 600 million read pairs from SB integration libraries [5], including reads that began with non-TA sequences as well as TA dinucleotides
Summary
Sleeping Beauty transposon (SB) has become an increasingly important genetic tool for generating mutations in vertebrate cells. It is widely thought that SB exclusively integrates into TA dinucleotides This strict TA-preference has not been rigorously tested in large numbers of insertion sites that can be detected with generation sequencing. The Sleeping Beauty transposon (SB) is a DNA transposon of the Tc1/mariner family, which was constructed from a consensus transposable element sequence in the genome of the Salmonid subfamily of fish [1]. SB is capable of transposing in mammalian systems and has become a popular genetic tool for generating genome-wide mutations [2,3,4,5]. DNA transposons often have strong preferences for their target sites. In 2005, Yant et al identified more than 1300 SB insertions, which all targeted to TA dinucleotides [11], further confirming the strict TA
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