BINNER»SNP UPLOAD DETAILS

Binning Rule 1: Each FlexMAP "tag" can only occur once in a multiplexed assay

• FlexMAP "tags" are assigned to SNP probe sequences with the TagIT software (see also information on the FlexMap microspheres).
• SNP probes are synthesised with the "FlexMAP tag" sequence at their 5' end.
• For each SNPmarker, the "FlexMAP tags" (abbreviated as "LUA (LUminex Array) number", e.g. "LUA19" refers to the "FlexMAP tag" with zip code number "19") of the two associated SNP probes are imported into GMT under headings SNP1LUA and SNP2LUA in the ADMIN»SNP MARKER DB IMPORT file.
• Using this information, SNPBINNER ensures that in a given well, each "FlexMAP tag" occurs only once. If no entries exist in these fields, a SNP marker is "rejected" from binning.

Reason for applying this rule:

• The "FlexMAP tag" sequence at the 5' end of a SNP probe is used to attach the probe to a microsphere carrying the reverse-complementary FlexMAP "anti-tag" oligonucleotides. Each set of uniquely coloured microspheres carries a different anti-tag sequence, allowing to relate a given SNP probe (and its associated reporter dye intensity) to a given, identifiable, set of microspheres.
• To date, 100 different "FlexMAP tags" exist, necessitating their repeated use when tagging a potentially unlimited number of possible SNP probes.
• When SNP probes are analysed together in multiplexed OLA or ASPE assays, it is therefore necessary to ensure that two probes carrying the same "tag" are not used together. If this happened, the two SNP probes with the same "tag" would bind to the same set of microspheres, making it impossible to relate the probe-related signal for that set of microspheres to either of the SNP probes.
• Please also refer to this "cartoon", illustrating the FlexMAP microsphere-based "Allele Specific Primer Extension" (ASPE) assay:

The FlexMAP microsphere-based "Allele Specific Primer Extension" (ASPE) assay.

1. Contigs are PCR-amplified from genomic DNA with "Locus-Specific Primers" ("FwdLSP" and "RevLSP").
   
2. For each SNP, two probes are designed. Each is a perfect match to one of the expected SNP alleles, with the nucleotide matching the polymorphic base at the 3' end.
   The 5' end of each probe carries a unique 24-base "FlexMAP tag", which is reverse-complementary to "FlexMAP anti-tags" uniquely assigned to different sets of microspheres (see 3 below).
   For the probe with a matching 3'-terminal nucleotide, primer extension occurs. Biotinylated dCTPs are incorporated into the newly polymerised DNA strand, allowing to label extended probes selectively with a streptavidin-linked reporter dye.
3. Probes are uniquely annealed to microspheres through the specificity of the FlexMAP "tag" / "anti-tag" recognition. The Luminex detection system identifies each microsphere by its internal dye, and records the associated reporter dye intensity (in the example "result table" as "Mean Fluorescence Intensity").
4. The reporter dye intensity detected for a pair of SNPprobes is used to calculate an "allele ratio" and interpreted as a genotype. In the illustrated example, the SNP's genotype is considered homozygous for allele "G".

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Binning Rule 2: Only one SNP on a given contig can be analysed in a multiplexed assay

• For each SNP marker, the contig name has to be entered under column heading contig in the ADMIN»SNP MARKER DB IMPORT file to allow SNPBINNER to apply this rule. If no contig entry is available, a marker is "rejected" from binning.

Reason for applying this rule:

• Occasionally, more than one SNP is found on a given contig. If probes for different SNPs on the same contig are used together in a primer extension reaction, it is possible that two SNP probes are placed in an orientation and distance from each other that allows a PCR product to be formed, with the two SNP probes acting as the flanking PCR primers. The resulting PCR product would have the "FlexMAP tags" from the two different probes at either end.
• When these PCR products are annealed to the microspheres carrying the reverse-complementary "FlexMAP antitags", they would link the two microspheres assigned to the two different SNP probes together, making it impossible to distinguish signals from the different SNP probes.

• Please also refer to this cartoon for an illustration:
  

  Microsphere cross-linking when more than one SNP is analysed on a given contig in a multiplexed assay

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Binning Rule 3: SNPs whose probes are known to form dimers are not multiplexed

• If it is known (e.g. from a BLAST search, comparing SNP probe sequences to other SNP probe sequences) that a SNP probe can anneal to other SNP probe(s), this information can be entered in field excludedprobes for a SNP marker in the ADMIN»SNP MARKER DB IMPORT file. Entries in this field are optional.
• SNPBINNER will use entries under "excludedprobes" to prevent that SNPs with cross-reacting probes are combined in a given well.

Reason for applying this rule:

• "Probe dimers" are undesirable for different reasons. One is that probes are effectively "removed" from the SNP assay when they form dimers. Another aspect is that a "probe dimer" could form a "link" between microspheres, making it impossible to distinguish between the fluorescence related to different SNP probes.

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Binning Rule 4: SNPs known to be amenable to probe / contig mispriming are not multiplexed

• If it is known (e.g. from a BLAST search, comparing SNP probe sequences to contig sequences) that a SNP probe can anneal to a "non target" contig and prime DNA strand extension, this information can be entered in field excludedcontigs for a SNP marker in the ADMIN»SNP MARKER DB IMPORT file. Entries in this field are optional.
• SNPBINNER will use entries under "excludedcontigs" to prevent that SNPs with cross-reacting probes / contigs are combined in a given well.

Reason for applying this rule:

• The fluorescence detected for a SNP probe extended after annealing to a "non target" contig does not serve to measure presence of the related SNP allele, but the presence of an unknown sequence on another contig.

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