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.
Smiraglia
Dominic J
DJ
Department of Cancer Genetics, and Comprehensive Cancer Center, Roswell Park Cancer Institute, Buffalo, NY, USA. dominic.smiraglia@roswellpark.org
Kazhiyur-Mannar
Ramakrishnan
R
Oakes
Christopher C
CC
Wu
Yue-Zhong
YZ
Liang
Ping
P
Ansari
Tahmina
T
Su
Jian
J
Rush
Laura J
LJ
Smith
Laura T
LT
Yu
Li
L
Liu
Chunhui
C
Dai
Zunyan
Z
Chen
Shih-Shih
SS
Wang
Shu-Huei
SH
Costello
Joseph
J
Ioshikhes
Ilya
I
Dawson
David W
DW
Hong
Jason S
JS
Teitell
Michael A
MA
Szafranek
Angela
A
Camoriano
Marta
M
Song
Fei
F
Elliott
Rosemary
R
Held
William
W
Trasler
Jacquetta M
JM
Plass
Christoph
C
Wenger
Rephael
R
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