Restriction landmark genomic scanning (RLGS) spot identification by second generation virtual RLGS in multiple genomes with multiple enzyme combinations.
BACKGROUND
Restriction landmark genomic scanning (RLGS) is one of the most successfully applied methods for the identification of aberrant CpG island hypermethylation in cancer, as well as the identification of tissue specific methylation of CpG islands. However, a limitation to the utility of this method has been the ability to assign specific genomic sequences to RLGS spots, a process commonly referred to as "RLGS spot cloning."


RESULTS
We report the development of a virtual RLGS method (vRLGS) that allows for RLGS spot identification in any sequenced genome and with any enzyme combination. We report significant improvements in predicting DNA fragment migration patterns by incorporating sequence information into the migration models, and demonstrate a median Euclidian distance between actual and predicted spot migration of 0.18 centimeters for the most complex human RLGS pattern. We report the confirmed identification of 795 human and 530 mouse RLGS spots for the most commonly used enzyme combinations. We also developed a method to filter the virtual spots to reduce the number of extra spots seen on a virtual profile for both the mouse and human genomes. We demonstrate use of this filter to simplify spot cloning and to assist in the identification of spots exhibiting tissue-specific methylation.


CONCLUSIONS
The new vRLGS system reported here is highly robust for the identification of novel RLGS spots. The migration models developed are not specific to the genome being studied or the enzyme combination being used, making this tool broadly applicable. The identification of hundreds of mouse and human RLGS spot loci confirms the strong bias of RLGS studies to focus on CpG islands and provides a valuable resource to rapidly study their methylation.
Department of Cancer Genetics, and Comprehensive Cancer Center, Roswell Park Cancer Institute, Buffalo, NY, USA. dominic.smiraglia@roswellpark.org
Dominic J
DJ
Smiraglia
Ramakrishnan
R
Kazhiyur-Mannar
Christopher C
CC
Oakes
Yue-Zhong
YZ
Wu
Ping
P
Liang
Tahmina
T
Ansari
Jian
J
Su
Laura J
LJ
Rush
Laura T
LT
Smith
Li
L
Yu
Chunhui
C
Liu
Zunyan
Z
Dai
Shih-Shih
SS
Chen
Shu-Huei
SH
Wang
Joseph
J
Costello
Ilya
I
Ioshikhes
David W
DW
Dawson
Jason S
JS
Hong
Michael A
MA
Teitell
Angela
A
Szafranek
Marta
M
Camoriano
Fei
F
Song
Rosemary
R
Elliott
William
W
Held
Jacquetta M
JM
Trasler
Christoph
C
Plass
Rephael
R
Wenger
D000818:Animals; D019295:Computational Biology; D018899:CpG Islands / Q000235:genetics*; D019175:DNA Methylation; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D016678:Genome / Q000235:genetics*; D015894:Genome, Human / Q000235:genetics; D023281:Genomics / Q000379:methods*; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D051379:Mice; D009928:Organ Specificity / Q000235:genetics; D015183:Restriction Mapping / Q000379:methods*
D004262:DNA Restriction Enzymes