HtStuf: High-Throughput Sequencing to Locate Unknown DNA Junction Fragments

Advances in high-throughput sequencing have led to many new technologies for assessing genomes and population diversity. In spite of this, inexpensive and technically simple methods for efficiently pinpointing the location of transgenes and other specific sequences in large genomes are lacking. Here we report the development of a modified TA cloning and Illumina sequencing method called high-throughput sequencing to locate unmapped DNA fragments (HtStuf). Transgenic insertion sites were identified and confirmed in nine out of 10 transgenic soybean [Glycine max (L.) Merr.] lines, and major rearrangements of the transgene were detected in these lines. Additionally this method was used to map insertions of the introduced DNA transposon, mPing, in four T6 lines derived from a single event. Fifteen of the mPing insertion sites were validated with polymerase chain reaction. Together, these data demonstrate the simplicity and effectiveness of this novel sequencing method.