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When you use the Ion AmpliSeq Designer, the tiling and pooling algorithms run behind the scenes to transform your submitted design into an orderable Custom AmpliSeq kit.
The following sections describe the AmpliSeq tiling and pooling processes:
The pipeline is optimized to find the highest coverage with the least number of amplicon pools. In an attempt to exhaustively design all primer pairs, the pipeline creates an exhaustive search by sliding a tiling window one-base at a time across the entire targeted region.
In creating the primers, the pipeline takes in consideration:
A biological filtering
An off-target analysis to minimize off-target hybridization
The tiling algorithm selects a subset (a tiling) of the input amplicons that meets the following criteria:
To do this, the tiling algorithm performs the following steps:
The output from the tiling algorithm is used as input for the pooling algorithm.
Improvements in the tiling algorithm have significantly increased the in-silico coverage of the designs. The users are enabled to choose between in-silico and assay performance.
The pooling algorithm assigns each tile (amplicon) to a pool, subject to requirements that allow each pool to be multiplexed (see "Pooling Requirements", below).
To assign each tile to a pool, the pooling algorithm performs the following steps:
The following requirements must be met for the pooling algorithm to assign a tile to a pool.
A pool is a set of amplicons meant to be multiplexed (amplified together in a single tube).
The minimum balance factor for every pool p is:
The minimum amplicon separation is 50 bp.