Custom multiplex tiling PCR panel design tool

Custom multiplex tiling PCR panel design for amplicon sequencing technology, targeting next-generation (NGS) and third-generation (TGS) DNA sequencing technologies. Multiplex amplicon sequencing is a powerful method suitable for targeted sequencing across a wide range of organisms, including humans, plants, animals, and microorganisms - assuming that reference genome sequences are available. This tool enables users to design custom multiplex tiling PCR panels optimized for high-throughput amplicon sequencing to identify genetic variation in natural or mutagenized populations through eco-tilling or multiplex CRISPR screens. It ensures high primer compatibility within each panel and comprehensive target region coverage.
Tiling Strategy: The tool will divide the target region into overlapping amplicons, ensuring full coverage. Two complementary primer panels (A and B) are automatically generated. All primer pairs within each panel are designed to be mutually compatible for simultaneous multiplex PCR reactions, minimizing primer-dimer formation and cross-reactivity.
Input format: Sequences can be pasted or uploaded as a file in FASTA format or retrieved sequence (NCBI’s accession, e.g. A02710) from NCBI’s "nuccore" nucleotide database. This approach is particularly effective for capturing genomic regions smaller than 1 megabase (Mb) and is compatible with both short-read (Illumina) and long-read (Oxford Nanopore Technologies) sequencing platforms.
Desired amplicon size range, which may vary depending on the sequencing platform: For short-read NGS (Illumina), typical amplicons range from 150-400 bp. For long-read TGS, larger amplicons range from 500-3,000 bp or more may be used.
Output: Downloadable list of designed primers with detailed information: sequences, genomic coordinates, amplicon sizes, and melting temperatures. Ready-to-use primer mixes for synthesis or experimental validation.

Primer design option
Length range (12-100 nt):	-
Tm range (°C):	-
Minimal linguistic complexity (70-90%):
Variants of the 3'-end composition (5'-3'):
Non-specific priming control	Overlaping primers
	C>>T bisulfite conversion
Range of PCR product size (bp):	-
The gap between amplicons (bp):
Forward primer tail (5'-3'):
Reverse primer tail (5'-3'):
Generate		Clear

To export the results: select all (Ctrl-A), copy (Ctrl-C) and paste (Ctrl-V) to Excel sheet.

Sequences are expected to be represented in the standard IUB/IUPAC nucleic acid codes are acceptable letters: B=(C,G,T), D=(A,G,T), H=(A,C,T), K=(G,T), M=(A,C), N=(A,C,G,T), R=(A,G), S=(G,C), V=(A,C,G), W=(A,T), Y=(C,T), U=Uracil, I=Inosine.

Variants of the 3'-end composition (5'-3'): the structure of the last nucleotides at the 3'-end of the primer, it can be specified by "N" for any pattern, or it can be encoded by one, two, three or more characters of standard or mixed letters. It is possible to specify one or more patterns (separated by spaces and of equal length): sws ssw sww wss www. For example, the pattern WSS corresponds to all variants of the 3'-end composition: acc acg agc agg tcc tcg tgc tgg.

Pre-designed list of primers/probes. Pre-designed primers/probes list is used for multiplexing with prior designed PCR primer/probe sets.

Linguistic sequence complexity (LC%) is a measure of the 'vocabulary richness' of a genetic text, based on counting the number of possible nucleotide combinations ('entropy' of the set of possibilities) relative to the theoretical maximum. This sequence value is converted into a percentage, with 100% representing the highest possible level.

Optionally, use two ‘/.../’ signs for the start and end of the excluded region (this is possible multiple times).

A single target sequence is allowed, either for the entire length or within the limiters (‘[‘ and ‘]’ ), or multiple target fragments within the sequence:
Single target:   [ Single target ]
Miltiple targets:   [ target 1 ]  [ target 2 ]   [ target 3 ] ....

С >> T bisulfite conversion (bisulfite modified genome)

Sequence, design of specific PCR primers for in silico bisulphite conversion for both strands - only cytosines not followed by guanidine (CpG methylation) will be replaced by thymines:

5’aaCGaagtCCCCa-3'        5’aaCGaagtTTTTa-3'
  |||||||||||||     ->      ||||||:|::::| 
3’ttGCttCaggggt-5'        3’ttGCttTaggggt

Non-specific priming control

Oligonucleotide specificity is one of the most critical factors for good PCR; optimal primers should hybridize only to the target sequence, especially when using complex genomic DNA as a template. Amplification problems can occur when primers anneal to repetitive sequences (retrotransposons, transposons or inverted tandem repeats). Alternative product amplification can also happen when primers are complementary to inverted repeats and produce multiple bands. However, the generation of inverted repeat sequences is exploited in two common generic DNA fingerprinting methods (RAPD).