- AnthOligo streamlines the arduous process of creating internal oligos,
cross-comparing and filtering them via complex algorithm to create a final optimal set of oligos.
- Multiple regions of interest (ROI) can be analyzed together.
- Configure button toggles custom parameters for users to set for each task. Suggested range of values are provided in parenthesis next to each label.
- All ROIs in the file are processed and analyzed simultaneously, such that it generates one optimal set of oligos for every ROI.
Oligos from each region are also cross-compared against oligos from every other region.
- Multiple ROIs may be analyzed together on the wet-bench, hence, analyzing them together will help filter any oligos that dimerize across ROIs.
- This results in unique combination of sets of oligos that would not lead to unnecessary cross-amplifications.
Complexity of the algorithm may result in limitations on your input ROI to be limited to a cumulative size of 1MB. This is only because of the resource restrictions on our production environment. However, if a user wishes to submit regions whose cumulative input ROI size is larger than this provided limit, please contact DIMITRI MONOS (monosd at email.chop.edu) and we will be happy to run it for you on our internal AnthOligo.
CONFIGURE button toggle: Due to our web server configuration, the configure button toggle dependency scripts are not tied to https which is why you may see this warning by browser. In case of CONFIGURE button toggle issues, please try one of the two options below to view the parameters.
Google Chrome browser users - If the CONFIGURE toggle button does not expand, try one of the two options: click on the little shield icon in the URL pane and select "Load Unsafe Scripts" to enable JavaScript OR click on the lock icon on the URL pane, click on site settings, scroll down to the last option, "Insecure content" and select "Allow" from the dropdown options. Close the settings page and reload the AnthOligo URL or click "Reload" on the already opened AnthOligo page when prompted to do so. This should enable JavaScript.Firefox browser users - If the CONFIGURE toggle button does not expand, click on the lock icon in the URL pane, click on the ">" button to the right of the "Connection secure" section to see connection details and select "Disable protection for now" to enable JavaScript.
Code Logic
AnthOligo implements multithreading for faster processing and efficient implementation of resources.
The algorithm processes multiple ROIs in parallel to produce results that are then cross-compared across all ROIs.
Here are some key points to note:
- The user submits a job to a task queue that processes jobs on a first-in-first-out (FIFO) principle
- AnthOligo implements a moving window approach to model selection of the most optimal set of internal oligos
- Calculations can get tedious! A Redis-like implementation helps store really large datasets in memory overflowing to disk.
- Multiple Graph-based data-structures help define sets of oligo sets.
- Your final result will include one optimal set of oligos for each ROI. This optimal set of oligos consists of oligos
that work well with each other and also do not react with final oligo sets in other ROIs (if provided in the same batch)
Being an NP-hard problem, the final set of oligos are randomly picked if there is more than one set of optimal results.
Running AnthOligo more than one time, may give you multiple different combinations of optimal oligos if many results exist for an ROI
Output File format Help:
The output file is tab-delimited and contains the following data for each query region. Here is an explanation of headers:
| Header |
Description |
|
Primer Set
|
A unique id for a list of oligos for a query region.
|
|
Primer Id
|
A unique id for a for a specific oligo.
|
|
PrimerChr
|
Oligo chromosome coordinate on the selected assembly.
|
|
Primer Start
|
Oligo start position on the selected assembly.
|
|
Primer End
|
Oligo end position on the selected assembly.
|
|
Sequence
|
Oligo sequence.
|
|
Sequence Rev. Complement
|
Reverse complement of the oligo sequence.
|
|
GC
|
Percent GC (%) in sequence - from Primer3.
|
|
Tm
|
Melting temperature (C) of oligo sequence - from Primer3.
|
|
Size
|
Oligo sequence length.
|
|
Self Dimer
|
Oligo Sequence self-dimer free energy in kCal/mol.
|
|
Hairpin dG
|
Free energy for oligo sequence hairpin in kCal/mol.
|
|
Blat
|
Number of BLAT hits for the specific oligo sequence.
|
Example format (Hover over the headers to see more information)
Your input file needs to be tab delimited text file with three columns:
| Chromosome |
Start |
End |
| chr2 |
159168697 |
159180688 |
| chr5 |
145026057 |
145066304 |
| chr8 |
142633801 |
142664168 |