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For a custom HaloPlex design, the SureDesign probe selection process avoids probes that cover repetitive sequences or sequences that may be difficult to capture due to known genetic variations. The process also favors selecting probes at a sufficient density to promote the capture of previously unknown genetic variations.
To avoid selecting probes that may capture non-specific targets, SureDesign uses a K-mer database filter to discard probes in which both probe arms cover a repetitive region. SureDesign does not automatically discard probes that have only one probe arm in a repetitive region.
To avoid selecting probes that may not capture the intended target due to genomic variability, SureDesign uses the NCBI dbSNP database to identify the locations of annotated insertions, deletions, and single nucleotide polymorphisms (SNPs) in the target regions. SureDesign discards probes that meet any of the following criteria:
· The probe covers a region containing an annotated insertion or deletion
· The restriction enzyme recognition sequence within the probe covers an annotated SNP location
· The SNP covered in the probe could introduce a new restriction enzyme recognition sequence
For typical exon-capture designs, SureDesign selects probes such that target nucleotides are covered an average of 4 probes. This level of coverage helps promote the capture of sequences that may contain novel genetic variations.