Restriction landmark genomic scanning (RLGS) spot identification by second generation virtual RLGS in multiple genomes with multiple enzyme combinations.

BackgroundRestriction 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."ResultsWe 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.ConclusionThe 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

Restriction Landmark Genomic Scanning (RLGS) spot identification by second generation virtual RLGS in multiple genomes with multiple enzyme combinations-6

<p><b>Copyright information:</b></p><p>Taken from "Restriction Landmark Genomic Scanning (RLGS) spot identification by second generation virtual RLGS in multiple genomes with multiple enzyme combinations"</p><p>http://www.biomedcentral.com/1471-2164/8/446</p><p>BMC Genomics 2007;8():446-446.</p><p>Published online 30 Nov 2007</p><p>PMCID:PMC2235865.</p><p></p>er interface along with the human master profile. Three veiwports in the Conime interface. The master profile is seen in the bottom left and the virtual profile is shown on the bottom right. The upper panel is a composite image of the two with the virtual profile contributing red and the master profile contributing blue. Areas of spot overlap between the two profiles appear as dark blue. Zoomed in view of the gels in A). Spots 38, 45, 46, and 48 are known sequences and are labeled on both the master profile and virtual profile with their overlap shown in the upper panel. The arrow in all three profiles indicates the spot of interest on the master profile, the top candidate sequence on the virtual profile, and their overlap. To the right is shown a window that can be selected to provide information about the virtual spot of interest (clicking on the spot with the arrow). This spot of interest is found at chr16:86976875

Restriction Landmark Genomic Scanning (RLGS) spot identification by second generation virtual RLGS in multiple genomes with multiple enzyme combinations-3

<p><b>Copyright information:</b></p><p>Taken from "Restriction Landmark Genomic Scanning (RLGS) spot identification by second generation virtual RLGS in multiple genomes with multiple enzyme combinations"</p><p>http://www.biomedcentral.com/1471-2164/8/446</p><p>BMC Genomics 2007;8():446-446.</p><p>Published online 30 Nov 2007</p><p>PMCID:PMC2235865.</p><p></p> overlap of the actual and virtual spots of interest – 62 and 68 – circled. All other labeled spots were previously identified. The vRLGS image with the extra spot filtering . Extra spots are indicated with red Xs. Spot A is a spot of interest for which the presence of extra spots makes the selection of the best candidate sequence difficult. The same vRLGS image with the extra spot filtering . Faded red Xs indicate the positions the extra spot occupied in A), but are not seen with the filtering . The candidate sequence for spot of interest A becomes immediately obvious. Example of PCR candidate sequence confirmation. DNA was eluted from the spots indicate as template DNA for PCR using primers derived from the candidate sequence for the spots of interest. In the last two lanes, primers for the candidate sequence for spot 68 were used to amplify DNA eluted from spot 62 and spot 68. Only the when the DNA eluted from spot 68 was used as template was a band observed. This is confirmation that the sequence predicted by the vRLGS in A) and B) is correct for spot 68. The same is true for spots 36 and 56 (not shown in A) and B)), and 62

Restriction Landmark Genomic Scanning (RLGS) spot identification by second generation virtual RLGS in multiple genomes with multiple enzyme combinations-5

<p><b>Copyright information:</b></p><p>Taken from "Restriction Landmark Genomic Scanning (RLGS) spot identification by second generation virtual RLGS in multiple genomes with multiple enzyme combinations"</p><p>http://www.biomedcentral.com/1471-2164/8/446</p><p>BMC Genomics 2007;8():446-446.</p><p>Published online 30 Nov 2007</p><p>PMCID:PMC2235865.</p><p></p>est, previously mapped in BXD recombinant inbred strains are labeled on the actual gel and candidate vRLGS spots are labeled that are located in the same genomic region as the genetically mapped spots. The circles in the upper panel indicate the overlap between the actual and virtual spots of interest. Same as in A) except using the enzyme combination NotI-PvuII-PstI. The spots of interest are genetically mapped in a BSS interspecific backcross

Restriction Landmark Genomic Scanning (RLGS) spot identification by second generation virtual RLGS in multiple genomes with multiple enzyme combinations-1

<p><b>Copyright information:</b></p><p>Taken from "Restriction Landmark Genomic Scanning (RLGS) spot identification by second generation virtual RLGS in multiple genomes with multiple enzyme combinations"</p><p>http://www.biomedcentral.com/1471-2164/8/446</p><p>BMC Genomics 2007;8():446-446.</p><p>Published online 30 Nov 2007</p><p>PMCID:PMC2235865.</p><p></p>�7 are known sequences and are labeled on both the master and virtual profiles, with their overlap shown in the upper panels. The virtual spot labels are grey and outlined in red. show the same window of the gels using the indicated migration formulas for the first and second dimensions (see materials and methods for formulas)

Restriction Landmark Genomic Scanning (RLGS) spot identification by second generation virtual RLGS in multiple genomes with multiple enzyme combinations-4

<p><b>Copyright information:</b></p><p>Taken from "Restriction Landmark Genomic Scanning (RLGS) spot identification by second generation virtual RLGS in multiple genomes with multiple enzyme combinations"</p><p>http://www.biomedcentral.com/1471-2164/8/446</p><p>BMC Genomics 2007;8():446-446.</p><p>Published online 30 Nov 2007</p><p>PMCID:PMC2235865.</p><p></p>t absent from the liver profile. vRLGS images of the same region with the extra spot filtering turned in the first panel, but turned in the second panel. The third panel shows the overlay of the mouse master profile where the spot of interest is not seen and the vRLGS profile with the extra spot filtering turned . The intestine specific spot of interest is only seen on the vRLGS profile in the extra spot filtering is turned . This candidate sequence from the vRLGS profile was confirmed to be correct by performing a BAC clone mixing gels shown in the third panel of A) where the spot of interest is greatly enhanced by the radio-labeled BAC

Restriction Landmark Genomic Scanning (RLGS) spot identification by second generation virtual RLGS in multiple genomes with multiple enzyme combinations-2

<p><b>Copyright information:</b></p><p>Taken from "Restriction Landmark Genomic Scanning (RLGS) spot identification by second generation virtual RLGS in multiple genomes with multiple enzyme combinations"</p><p>http://www.biomedcentral.com/1471-2164/8/446</p><p>BMC Genomics 2007;8():446-446.</p><p>Published online 30 Nov 2007</p><p>PMCID:PMC2235865.</p><p></p> charts, data for the extra spots is shown in pink and data for real spots is shown in blue. The Y axes for all are the percentage of the class of spots showing the measure indicated on the X axis Measures of GC content. Observed/expected CpG ratio. Number of CpGs. The percentage of spots retain after eliminating all spots that fail to meet the three indicated criteria. The boxed data points show the percentage of real and extra spots retained when all spots that fail to meet all three of the following criteria are eliminate: 50

Restriction Landmark Genomic Scanning (RLGS) spot identification by second generation virtual RLGS in multiple genomes with multiple enzyme combinations-0

<p><b>Copyright information:</b></p><p>Taken from "Restriction Landmark Genomic Scanning (RLGS) spot identification by second generation virtual RLGS in multiple genomes with multiple enzyme combinations"</p><p>http://www.biomedcentral.com/1471-2164/8/446</p><p>BMC Genomics 2007;8():446-446.</p><p>Published online 30 Nov 2007</p><p>PMCID:PMC2235865.</p><p></p>er interface along with the human master profile. Three veiwports in the Conime interface. The master profile is seen in the bottom left and the virtual profile is shown on the bottom right. The upper panel is a composite image of the two with the virtual profile contributing red and the master profile contributing blue. Areas of spot overlap between the two profiles appear as dark blue. Zoomed in view of the gels in A). Spots 38, 45, 46, and 48 are known sequences and are labeled on both the master profile and virtual profile with their overlap shown in the upper panel. The arrow in all three profiles indicates the spot of interest on the master profile, the top candidate sequence on the virtual profile, and their overlap. To the right is shown a window that can be selected to provide information about the virtual spot of interest (clicking on the spot with the arrow). This spot of interest is found at chr16:86976875