Monday, June 12, 2017

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Annotation by MSU students

Tentative identification of tac sites by the BIOL 497 Genomics class at MSU

Annotated tac/tds Table (by MSU students)  

The objective of the BIOL 497 Genomics class at MSU was to identify putative tac sites among the SOLiD contigs on the basis of contig coverage and location, as all the Acs in the collection had been previously mapped relative to the wx donor locus.  The classes were taken in the spring of 2010 and 2011 by fourteen and fifteen undergraduate students, respectively.  Students were assigned chromosomes or parts of chromosomes, with chromosome 9 assigned to two students because the control in the first pool of 96 mapped there.  Graduate student Andrew Hoffman was the teaching assistant for this course and checked the primers designed by the undergraduate students.


Students BLASTed sequences against the htgs maize sequences in GenBank to obtain the locations of contigs.  They, then, searched 5 kb of flanking regions for the terminal inverted repeats (TIRs) of Ac and Ds.  Contigs containing TIRs were excluded, as these are most likely Ds elements shared by W22 and B73.  Among the remaining contigs, those found in or near (<400 bp) genes were considered as potential tac sites because Ac inserts preferentially within genes (Cowperthwaite et al. 2002).  Only those sequences present in one or two copies were used for primer design.


A total of 150 primers were designed by the students and double-checked by TA Hoffman.  A group of three students, African-American Onsarigo Nyabeta (who is a master student at MSU now), Laura E. Simone (who is working on her Ph.D. at Duke Medical School currently), and Daniella R. Menillo, along with the class TA, carried out pilot PCR verification of tac sites in the Dooner lab in early May under the supervision of Limei He, the Dooner lab researcher.  The results are promising.  Twelve primers, corresponding to 12 unknown tac sites from chromsome 9 and 1 positive control, were tested on the pool of 96 DNAs, and 3 putative new tac sites were identified, which are sequenced by Limei He.


The detailed annotation results can be found at our ‘Annotated tac/tds Table’.


Annotation of Ac/Ds distribution in B73 and W22 maize inbred lines via undergraduate independent research


Five undergraduates earned independent research credits in Fall 2009 and Spring 2010 by assisting M.S. student Andrew Hoffman in the annotation of Ac/Ds transposable elements in the B73 genome and in the analysis of the subset shared by W22.  The results of this analysis are being prepared for publication.  Using an algorithm to search through the maize pseudomolecules, about 1000 Ds-like elements were located and annotated.  There are 367 type 1 Ds1 elements and 635 type 2 Ac deletion derivatives.  Among the type 2 Ds elements, 36 are related to the 1.3-kb Ds2, 36 have homology to the internal transposase of Ac, 352 are composed of only truncated subterminal regions of Ac, and the rest share similarity with only the 3’ end of Ac.  Forty-six type 1 and one hundred seventy-three type 2 Ds elements appear to have inserted into genes. The size of the type 2 elements ranges from 600 bp to 20 kb. Helitrons, LTR retrotransposons, and full-length cDNAs can be found embedded in the larger elements.  We are also using next-generation sequencing technology to characterize the spectrum of Ds-like elements in W22.