TILLING® and EcoTILLING® with JelMarker™ Image Reader and Conversion Software. The Mutation Detection module within JelMarker rapidly identifies SNPs in TILLING and EcoTILLING data with a new trace-to-trace comparison technology that visually displays SNPs in an easy to read electropherogram trace. The module also includes a unique algorithm for sizing large TILLING fragments beyond 700 basepairs. JelMarker's trace-to-trace physical comparison and unique sizing algorithm make it ideal software for researchers detecting SNPs in TILLING and EcoTILLING gel images.
- Exclusive "Physical Comparison" quickly detects SNPs even those not discernable by visual inspection
- Low false positive rate
- Unique sizing algorithm for larger fragments
- High Sensitivity reduces costs by allowing pooling of samples
- Accepts image data from all major gel electrophoresis systems
- Easy-to-Learn and use
Targeting Induced Local Lesions In Genomes - or TILLING - was first introduced in 2000, through the Arabidopsis genome project. The TILLING technique uses chemical mutagenesis with ethylmethanesulfonate (EMS) to induce point mutations throughout an entire genome. The mutagenized founder population is crossbred and the resultant population is analyzed for gene specific point mutations. EcoTILLING takes regular TILLING one step further in that it allows for haplotyping groups or species based on natural allele variation identified through SNP discovery.
Visual Detection of SNPs in a Gel Image
In the TILLING image on the left, the first SNP fragment is detected in the 700 blue dye channel at position y=1740 pixels. In the image on the right, the second SNP fragment is detected in the green 800 dye channel at position y=3985 pixels.
SNPs are detected by comparing the two dye color gel images. The LI-COR IRDye® 700 - blue-labeled image will show a mutation fragment band just below the wildtype band. The counterpart band will be visible in a location exactly opposite of it on the LI-COR IRDye® 800 - green-labeled image. This fragment in the 800 dye-labeled image is the complementary cleavage product of the 700 dye-labeled image band. The sum of the length of these two fragments is equal to the original amplicon, which makes it easy to distinguish from PCR artifacts. This step can be performed visually with GelBuddy© software. JelMarker takes it one step further to detect even the faintest fragments automatically!
Reduction in False Negative SNP Calls
In the Show Mutations window, the blue peak is at 630bps and the green peak is at 820bps. These SNP fragments were missed with visual image analysis.
Converting the gel image to a trace view and performing a physical trace comparison significantly reduces the amount of time an analyst has to spend identifying small fragments on a gel image. Converting to traces also allows for detection of less intense bands that the human eye may miss resulting in a false negative call. This is especially important when analyzing EcoTILLING data which contains one mutated portion out of 12 pooled samples. The SNP fragment in this case would be extremely faint.
Differential Mobility of Large Fragments is Accounted For
The Edit Size window on the left depicts JelMarker's unique size extension algorithm for fragments larger than 700bp. The image on the right is the Show Mutations window. Notice how the bp size of the blue and green peaks add up to 1480 basepairs (with a resolution of approximately 20 basepairs) as expected.
With JelMarker's unique Extend Size algorithm, the differential mobility of large DNA fragments is accounted for. In the example above, the ladder contains the original basepair sizing from LI-COR's IRDye 700 Ladder up to size 700bp. From 700bp to 1500bp, the Extend Size feature in the Edit Size module interpolates the mobility shift of the large peaks in TILLING data. From those interpolated sizes, an approximate size can be deduced with a resolution of less than 30 basepairs. Notice the basepair size ladder at the top of the Mutation trace.
JelMarker was developed in response to a growing demand for software that can analyze fluorescence, chemiluminescence and autoradiography gel image files - especially those from LI-COR®'s 4300 DNA Analyzer, KODAK®'s Image Station 4000R and Hitachi's FMBIO® II System. JelMarker can import up to two TIFF, BIP, GEL, JPEG, BMP, IMG and TXT files for comparison analysis. The software also exports trace files for easy upload to the fragment analysis software - GeneMarker®.
- TILLING Application Note [PDF]
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