Oli2go is a fully automated, accurate software tool to design specific primers and probes for multiplex applications. The following sections provide information about the use of the software, the basic workflow, input parameters and finally the output of oli2go.
- Citing oli2go
- File preparation
- Primer and probe design
- Probe specificity check
If you can use our tool, please consider citing our latest publication:
Hendling, M., Pabinger, S., Peters, K., Wolff, N., Conzemius, R., & Barišić, I. (2018). Oli2go: an automated multiplex oligonucleotide design tool. Nucleic acids research.
As oli2go was primarily designed to help users to design optimized probes and primers, we are constantly working on further improvements of this tool. Please contact us if you have any questions, recommendations or feedback. You can also write us an e-mail if your job was aborted or crashed. Just tell us your job id and we will find the source of error and might help you finding better parameters for the design. You can reach us via e-mail: email@example.com
Oli2go should give users the possibility to design specific primers and probes for multiplex assays in one step. Using more than one software tool for multiplex oligonucleotide design can be very exhausting and error-prone. For this, we developed an all-in-one tool which works fully automatically and highly accurate. Accurate means on one hand, that especially the specificity of the oligonucleotides is checked using a high number of background sequences (~ 100 million sequences). As a result, probes and primers tend less to unspecific results in the lab. On the other hand, the software is highly accurate concerning cross dimerisaiton. This means, only primers without any cross dimerisation are accepted. Therefore, the risk of unwanted amplification is reduced.
However, the user should always keep in mind, that this software only fulfils in silico design. This means, that primers and probes are optimized for the use in the lab concerning important design factors (specifcity, dimerisation, temperature,...). But there is still no insurance, that the resulting oligonucleotides are working the same way in the laboratory.
Oli2go is a software tool defined by a specific series of design steps, which play an important role for multiplex oligonucleotide design. The steps are as follows:
This step prepares necessary files for further the design process.
The input sequences will be divided into k-mers, whereas k is in the range of the pre-defined minimum and maximum primer and probe length. Afterwards, the temperature and hairpin formation will be checked of each k-mer. For melting temperature calculation and hairpin check please refer to the related chapters in this documentation.
Hybridisation and ligation probes should be specific concerning their target genes. The resulting probes of the previous step, are checked for specificity using standalone BLAST+ (version 2.7.0). This step needs high computational power and takes longer than the other steps, as the aim of this step is to return only highly specific probes. For this, the probes will be checked against custom BLAST databases derived from the Genbank, Genomes and WGS directories of NCBI's FTP server. The databases comprise around 100 million sequences from the following data:
- Whole Genome Shotgun
Users can choose which of the databases are used for specificity check. It is recommended to make sure that the input sequences can be found in the selected target sequences.
The task of this step is to find possible forward and reverse primers of the specific probes, which are in the defined product size range and do not form secondary structures with each other. We experienced high primer specificity concerning their target gene, if they are designed around the high specific probe.
Most amplification methods has to deal with the problem of human background DNA. Therefore, we implemented a primer specificity check against the Human Genome. For this, the Burros-Wheeler Aligner BWA is used to align the primers against the human genome.
The main problem of multiplex reactions are cross reactions between primers of different genes. This may lead to a high number of unspecific bindings, which badly influence the outcome. Therefore, we implemented a cross dimerisation check, which uses primer3's thermodynamic alignment tool to check all primers against each other for secondary structure formation. Only primers which primer dimer melting temperatures and deltaG values are below the defined parameters are taken into account for the final assay.
- Default parameters
- Sequence Input
- Primer and probe lengths
- Primer and probe melting temperature
- Product size
- Minimum product size difference
- Monovalent cations
- Divalent cations
- Primer/probe concentration
- Databases for probe specificity check
- Primer/probe hairpin Tm threshold
- Primer/probe hairpin deltaG threshold
- Cross dimer Tm threshold
- Cross dimer deltaG threshold
Default parameters for Microarray probe design, LNC3 probe design and Padlock probe design can be chosen by the user.
Users can choose between two types of probes. First probe design can be specified for conventional hybridization-based probes. Second, users can choose to design ligation-based probes, where the probe consists of two-part oligonucleotides. Design can also be limited to microarray probe design. were only probes will be designed and checked for their specificity using the selected background database(s).
Sequence input can be accomplished by manual input or file upload of sequence data in FASTA format. A minimum of two sequences should be provided, as oli2go is a tool for multiplex oligonucleotide design. Sequences must be provided in FASTA format. Ambiguous nucleotides are accepted by the software. However, the user should be aware that ambiguous nucleotides may reduce primer and probe specificity and increase the design complexity. This may also result in higher computing time. We recommend not to use more than 5% ambiguous nucleotides in your input.
Primer and probe lengths can be freely chosen by the user. For this, minimum and maximum number of base pairs (bp) need to be provided.
Primer and probe melting temperatures can be freely chosen by the user. For this, minimum and maximum temperature (°C) need to be provided.
Product size can be freely chosen by the user. For this, minimum and maximum length (bp) need to be provided. The product size is determined by the length of the sequence enclosed by forward and reverse primer.
A minimum difference between the product sizes can be selected by the user.
The concentration of monovalent salt cations (mM) in the PCR. This value is used for melting temperature calculations as well as dimerisation checks.
The concentration of divalent salt cations (mM) in the PCR. This value is used for melting temperature calculations as well as dimerisation checks.
The concentration of all deoxyribonucleotide triphosphates (dntps) (mM) in the PCR. This value is used for melting temperature calculations as well as dimerisation checks.
The total annealing oligonucleotide concentration (nM) in the PCR. This value is used for melting temperature calculations as well as dimerisation checks.
Users must choose which databases are used for probe specificity check. At least one database needs to be chosen. It is recommended to make sure that the input sequences can be found in the selected target sequences.
Maximum allowed melting temperature for hairpin formation. Primer3 suggests secondary structures melting temperatures should be at least 10°C below primer/probe melting temperature.
Maximum allowed deltaG value for hairpin formation. The more negative the value the stronger the binding. Values below -10000 cal/mol are not suggested.
Maximum allowed melting temperature for cross dimerisation. Primer3 suggests secondary structures melting temperatures should be at least 10°C below the primer melting temperature.
Maximum allowed deltaG value for cross dimerisation. The more negative the value the stronger the binding. Values below -10000 cal/mol are not suggested.
- Melting temperature calculation
- Secondary structures
This sofware uses the same melting temperature calculation methods as suggested by primer3. The calculations are based on the method by Rychlik W, Spencer WJ and Rhoads RE within their paper "Optimization of the annealing temperature for DNA amplification in vitro". Furthermore, the table of thermodynamic parameters and salt corrections provided by SantaLucia's paper "A unified view of polymer, dumbbell and oligonucleotide DNA nearest-neighbor thermodynamics" are used.
Secondary structure prediction is based on primer3's standalone component called ntthal, first presented in the paper "Primer3—new capabilities and interfaces". This software uses thermodynamic alignment to predict the propensity of an oligonucleotide to form dimers and hairpins. Oli2go takes the output melting temperature and deltaG value of ntthal to evaluate the secondary structure formation. For the secondary structure melting temperature, we follow primer3's suggestion, which says that it should be 10 degrees below the oligonucleotide melting temperature at maximum. Concerning the Gibbs Free Energy of secondary structure formation, we experienced that it should not be smaller than -10000 cal/mol.
After submitting a job, a new window pops up showing the status of the submission. The main page also shows the link to this status page. The results, input sequences and parameters will be also found at this page. Users must save the link or at least the ID of the session to access the status and the results of their submitted jobs.
The status of the design process is represented graphically by a progress bar. Furthermore, the already processed steps, the currently running step and the subsequent steps are visualized within a list below the progress bar. The steps are ordered from top to bottom. The completed steps are visualized in green with a check mark. Currently running steps are marked in blue and a running circle. Steps, which are still waiting to be processed are marked in white. Aborted steps are shown in red with a cross. In this case, the user has to return to the beginning and restart the process. The reasons for software abort could be too strict input parameters, wrong input parameters or problems with the input sequences.
Jobs which are submitted while other jobs are running on the server will be queued to prevent server overload. The user has the possibility to wait till the submission gets processed or abort the submission by clicking the "Abort" button. It is only possible to abort queued jobs.
The result page shows a list of all primers and probes, their sequences, lengths, product sizes, melting temperatures, hairpin temperatures and hairpin deltaG values. Furthermore, the user has the possibility to check the primers using Primer BLAST and to check the probes using online nucleotide BLAST. For this, links are located in the last column of the result table. After clicking the link, Primer BLAST or BLAST will be opened automatically already involving the sequences to check. The user can download the result table as CSV. Furthermore, the sequences can be downloaded in FASTA format.