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The Plant Genome Abstract - Original Research

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


This article in TPG

  1. Vol. 8 No. 1
    unlockOPEN ACCESS
    Received: Oct 22, 2014
    Accepted: Dec 15, 2014
    Published: March 13, 2015

    * Corresponding author(s): lkanizay@gmail.com
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  1. Lisa B. Kanizay *c,
  2. Thomas B. Jacobsac,
  3. Kevin Gillespiec,
  4. Jade A. Newsomec,
  5. Brittany N. Spaidc and
  6. Wayne A. Parrottabc
  1. c Center for Applied Genetic Technologies, Univ. of Georgia, Athens, GA 30602
    a Institute for Plant Breeding, Genetics and Genomics, Univ. of Georgia, Athens, GA 30602
    b Dep. of Crop and Soil Sciences, Univ. of Georgia, Athens, GA 30602


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.

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