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5-Nucleotide Keys


We use nucleotide "keys" embedded within the amplification primers to differentiate multiple samples on a GS-FLX run without physically partitioning the picotiter plate. We currently use 5-nucleotide keys, although longer or shorter keys can also work. Each key should have at least two nucleotides different from any other key used in a run. This guideline is critical because it ensures the likelihood of sequencing errors in the key causing reads to be misassigned between samples becomes statistically negligible. A shorter key reserves more of the flow cycles for variable region sequencing rather than primer region sequencing. On the other hand, longer keys provide more possible keys while still not violating the guideline of having at least a two nucleotide difference.

The 5nt key should always be included at the beginning of the read. It is placed between the A-adaptor and the forward primer for forward sequencing and between the B-adaptor and the reverse primer for reverse sequencing. For example, a forward sequencing run using the A-adaptor with a key of GATCT and the 967F-PP primer would use a complete primer of:

5'-gcctccctcgcgccatcagGATCTcnacgcgaagaaccttanc-3'


Below is the list of 5-nucleotide keys we are currently using:

GATCT
ATCAG
ACACT
AGCTA
CACAC
ACAGA
AGATG
CACTG
CAGAG
CGCAG
CTGTG
GTGAG
TCATG
AGCAT
CAGCT
CATGT
CTGAT
CTGCA
GATGA
TACGC
ACTGC
GTCAC
CGTAC
TGCGT
CGACG
CTACT
TGACT
GACAG
ATGCT
TCGTC
TATAC
ACGAC
TGTAG
TCGAG
TAGTG
CGAGT
ATACG
ACTCG
TCTGT
TCGCT
TACAT
GACGT
CTCGT
CGTCT
TACTA
ACTAT
AGTGT
ATAGT
ACGCA
AGACA
ATATA
ATCGA
CAGTA
CGCGA
CTCTA
GTAGA
GTGTA
TAGCA
TCACA
TGATA
TGTCA
ACATC
AGAGC
ATGTC
CGCTC
CTAGC
GACTC
GAGAC
GCTAC
GTATC
TGCAC
TGTGC
CATCG
GAGCG
GCACG
GCGTG
GTCTG
TGCTG
GCAGT
GCGAT
GCATA
GCTCA


If you wish, you can download this information in a text file.

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