The DNA transposon piggyBac is widely used as a tool in mammalian experimental 
systems for transgenesis, mutagenesis, and genome engineering. We have 
characterized genome-wide insertion site preferences of piggyBac by sequencing a 
large set of integration sites arising from transposition from two separate 
genomic loci and a plasmid donor in mouse embryonic stem cells. We found that 
piggyBac preferentially integrates locally to the excision site when mobilized 
from a chromosomal location and identified other nonlocal regions of the genome 
with elevated insertion frequencies. piggyBac insertions were associated with 
expressed genes and markers of open chromatin structure and were excluded from 
heterochromatin. At the nucleotide level, piggyBac prefers to insert into 
TA-rich regions within a broader GC-rich context. We also found that piggyBac 
can insert into sites other than its known TTAA insertion site at a low 
frequency (2%). Such insertions introduce mismatches that are repaired with 
signatures of host cell repair pathways. Transposons could be mobilized from 
plasmids with the observed noncanonical flanking regions, indicating that 
piggyBac could generate point mutations in the genome.