The MRPrimerW server performs complete homology testing, supports batch design of primers for multi-target qPCR experiments, and ranks the resulting primers to return the top-1 best primers to the user. To ensure high accuracy, we adopted the core algorithm of a previously reported MapReduce-based method, MRPrimer, but completely redesigned it to allow users to receive query results quickly (typically, within a minute) in the web interface, without requiring a MapReduce cluster or a long computation. MRPrimerW provides primer design services and a complete set of 341,963,135 validated primers covering 99% of human and mouse genes.
We reorganized the complex MRPrimer algorithm, which consists of seven MapReduce rounds, into two parts: offline processing and online processing. We built index structures using the results of offline processing and loaded them into the MRPrimerW web server. Using these indices, the online processing stage can quickly design high-quality primers against a user-specified target, as in a Google keyword search.
We used human and mouse genes in the consensus coding sequence (CCDS) database . In the most up-to-date CCDS datasets, there are currently 31,394 human gene DNA sequences (Release 18, the last update was 12 May, 2015) and a total of 24,833 mouse gene DNA sequences (Release 19, the last update was 30 July 2015).
Offline processing by MRPrimerW takes as input a DNA sequence database and several filtering constraints, and yields as output all possible primers that satisfy both homology testing and given filtering constraints. For filtering constraints, MRPrimerW considers eight parameters for each primer and five parameters for each pair. Most of these constraints are checked during online processing. However, four parameters (primer length, melting temperature, GC content, and contiguous residue) in below table are checked during offline processing, because primers with values out of the appropriate range (e.g., primer length 10 bp) are non-functional in general; consequently, they do not need to be considered during online processing.
|Single filtering||primer length||17~27 bp|
|melting temperature (TM)||56~63℃|
|contiguous residue||< 5-mer|
|cross-reactivity||up to 2 mismatch
up to 4 mismatch at 5’
After offline processing, we create a set of indices based on the results, which are then loaded into the main memory of the web server for online processing. Eight indices are built: six partial annotation indices, one full annotation index, and one primer index. All indices follow the structure of a key–value database, in which each row is a pair of key and value.
Online processing consists of three steps that check the filtering constraints provided by the users and rank the primers to return the top-1 best primers. The first step takes the user query and uses the indices to retrieve all candidate primers containing the query. While extracting the candidates, MRPrimerW applies eight filtering constraints for each primer. The second step applies five filtering constraints for primer pairs. The final step calculates the penalty scores of the primers obtained in the previous step and sorts the primers according to their scores. Then, it returns the top-1 best primer, i.e., the primer with the lowest penalty score, for each target sequence.
|Covered genes||Number of Primers|
|Human (Homo sapiens)||31,376||165,923,450|
|Mouse (Mus musculus)||24,797||176,039,685|
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