BatchPrimer3 v1.0

A high throughput web application for PCR and sequencing primer design

 

BatchPrimer3 is a comprehensive web primer design program using Primer3 core program as a major primer design engine to design different types of PCR primers and sequencing primers in a high-through manner. BatchPrimer3 allows users to design several types of primers including generic primers, hybridization oligos, SSR primers together with SSR detection, and SNP genotyping primers (including single-base extension primers, allele-specific primers, and tetra-primers for tetra-primer ARMS PCR), as well as DNA sequencing primers. A batch input of large number of sequences and a tab-delimited result output greatly facilitates rapid primer design and ordering process.

The BatchPrimer3 paper is available in BMC Bioinformatics. The citation is:

Frank M You, Naxin Huo, Yong Qiang Gu, Ming-cheng Luo, Yaqin Ma, Dave Hane, Gerard R Lazo, Jan Dvorak and Olin D Anderson. BatchPrimer3: a high throughput web application for PCR and sequencing primer designi. BMC Bioinformatics 2008, 9:253, doi:10.1186/1471-2105-9-253 [PubMed] [BMC Bioinformatics]

Primer design servers

USDA-ARS, Albany Server 1

USDA-ARS, Albany Server 2

UC Davis Server

Primer design methods

In BatchPrimer3 1.0, several types of primer design are implemented:
  • Generic primer design: The same features as Primer3 Web (Rozen and Skaletsky, 2000) are provided to design pairs of primers based on any DNA sequences.
  • SNP flanking primer design: Design pairs of primers that flank SNP sites.
  • Single base extension (SBE) primer design: SBE is a technique for detecting a known SNP site. Design a primer which anneals immediately adjacent to the SNP. Two best SNP primers, one for each orientation (forward and reverse), are designed for user selection according to scores.
  • Allele-specific primer design: Designs primers that will specifically amplify one of the alleles. For each orientation (forward and reverse) two best allele primers can be selected. Primer3 core program is used to pick the allele flanking primers and the similar algorithm for SNP primer design is used to choose the best allele primers.
  • SNP flanking primer and single base extension primer design: Design pairs of primers that flank the SNP site and a SNP primer that neighbor or include the SNP nucleotide.
  • Allele-specific primer design: Designs primers that will specifically amplify one of the alleles. Two primers that flank the alleles are picked. For each orientation (forward and reverse) two best allele primers can be selected from up to four allele primers (two for each orientation). Primer3 core program is used to pick the allele flanking primers and the similar algorithm for SNP primer design is used to choose the best allele primers.
  • Tetra-primer ARMS PCR primers: Ye et al. (2001) proposed a simple, effective and economical SNP genotyping method based on AS primers called tetra-primer ARMS-PCR. This procedure adopts principles of the tetra-primer PCR method and the amplification refractory mutation system (ARMS). Four primers are required to amplify a larger fragment from template DNA containing the SNP and two smaller fragments representing each of the two AS products. Primers are designed in such a way that the amplicons of two alleles differ in sizes and can be resolved by agarose gel electrophoresis. To enhance the specificity of the reaction, in addition to the first mismatch at the 3' end of AS primers, an extra mismatch is also deliberately introduced at the third position from the 3' end of each of the two inner AS primers. From the primer design perspective, two sets of tetra-primers for any SNP can be designed theoretically according to AS primer orientation. BatchPrimer3 v1.0 implemented a batch module to easily design two sets of tetra-primers for a SNP.
  • SSR screening and primer design: SSR or microsatellite is a simple sequence repeat, which is a useful genetic marker. SSR primers are picked from the SSR-flanking regions. SSR motif screening varies in criteria of SSR definition, i.e., motif length and number of motif repeats or SSR length. Typically dinucleotide motifs to hexanucleotide motifs are detected with at least 12 nucleotides in length of SSRs. Since the different criteria of SSR screening will result in large difference in screening results, BatchPrimer3 provides flexible options to allow users to set the screening criteria. A regular expression was used to design an algorithm to detect the repeat motifs and number of repeats. The detected SSRs are masked as targets. Then Primer3 program picks the best pairs of primers flanking the targets.
  • Hybridization oligo design: Design hybridization oligo primers. This module is the same as in Primer3 Web.
  • Sequencing primers: DNA samples can be sequenced in either the forward or reverse direction, or both. Candidates of a forward sequencing primer are scanned starting from the 5' end of target sequence until a primer meeting the userís parameter settings with a high quality score (greater than or equal to 60) is found. The same procedure is applied to selection of reverse sequencing primer except the starting point of primer scanning is the 3' end of target sequence.

Sequence Input

Two ways are available to input sequences:
  1. Sequences can be copied and then pasted to a sequence text box. This approach has a maximum 256 kb size limit.
  2. A FASTA file can be uploaded to the server and the sequence size limitation only depends on Internet speed and server machine memory.
A FASTA format is acceptable in the BatchPrimer3 program. See example.
  • For SNP flanking primers or allele-specific primer design, the SNPs or alleles in the sequence must be masked using IUB/IUPAC nucleic acid code (G/C→S, A/T→W, G/A→R, T/C→Y, G/T→K, A/C→M), and the sequence file follows the NCBI dbSNP FASTA format. See example.
  • For generic primer and oligo primer desgin, the “[]” pair can be used to specify targets, the “{}” pair to specify included region, and the “<>” pair to specify excluded region. The program can recognize them and automatically determine the proper parameters to pick primers to flank targets, or exclude the specified regions or only include the specified region for primer design. See example.

Sequence pre-analysis

A utility tool for pre-analysis of input sequences is provided in BatchPrimer3 to help users to understand the basic statistics of input sequences, such as sequence length, GC content and their distributions in a whole set of input sequences. The information provides hints to adjust the parameter ranges of product size and GC content. See example.

Result output

The BatchPrimer3 program produces four parts of outputs:
  1. a main HTML page containing the primer design summary of all input sequences;
  2. an HTML table page listing all designed primers and primer properties (see example);
  3. a tab-delimited text file with the same contents in the HTML table page,
  4. and a detailed primer view page for each sequence with successfully designed primers (see example). A simple click on the links on the main HTML page or HTML table page will display the primer view. The primer list can be directly saved as a text file or an Excel file for further editing or primer ordering.