Seq4SNPs: new software for retrieval of multiple, accurately annotated DNA sequences, ready formatted for SNP assay design

Table 1 Formatting comparison for three medium-throughput genotyping platforms

Genotyping method	Taqman[22]	SNPstream[23, 26]	Sequenom[24]
Assay design software	File Builder Assay-by-Design	Autoprimer	iPLEX
URL	https://www2.appliedbiosystems.com/support/software/assaysbydesign/	http://www.autoprimer.com/	http://www.sequenom.com
Assay name	Letters, numbers, – (hyphen) only	Letters, numbers, – only	Letters, number, – only
Space	Tab	Space	Space
No. bases each side	200–600	200	200
Allele [A/G]	Common first	-	-
Additional information <other>	1 = 201 (assay position)	Masked sequence	-
Adjacent SNPs	N	IUPAC	Lower case
Masking	N	N	Lower case
Masked sequence	First sequence	Second sequence, not 25 bases either side of SNP	First sequence

The low-medium genotyping platforms Taqman^®, Beckman SNPstream^® and Sequenom iPlex™ come with assay design software packages that have different input requirements: each takes a file containing multiple SNP sequences, one per line; each line begins with a name and is followed by a sequence, separated by a space or tab, succeeded by either the position of the assay SNP (Taqman^®) or any masked sequence (SNPstream^®) or nothing (iPLEX™). Additional SNPs flanking the SNP to be assayed can also cause suboptimal assay performance if designed into an oligonucleotide binding site. Proprietary assay design software avoids additional variations, if the input sequences are suitably annotated by changing the variant nucleotide in the sequence to N, to the IUPAC code or to lower case. Basic sequence formats are summarised in the table, and follow the convention:
Name <space> sequence [SNP]sequence <space> <other>.
Examples for annotation of a synthetic SNP where the flanking sequence contains a masked sequence (repetitive element shown as NNNNNN/catgga distal to allele) and one adjacent SNP (N/Y/c before allele):
e.g. for Taqman:
SNP-01_rs14352008 ATGCANCAGG [A/G]ATGNNNNNNCAGG 1=11
e.g. for SNPstream:
SNP-01_rs14352008 ATGCAYCAGG [A/G]ATGCATGGACAGG ATGCAYCAGG [A/G]ATGNNNNNNCAGG
e.g. for Sequenom:
SNP-01_rs14352008 ATGCAcCAGG [A/G]ATGcatggaCAGG