As of March 2012 we are using the Bioo Scientific NEXTflex barcoded adapters for WGS sequencing libraries made by ourselves, (well me so far). The set we are currently using comprises 48 barcodes, so we can multiplex up to a 48-plex in one lane on the Illumina HiSeq sequencer.
Bioo Sci. 48 barcoded adapters
Below are the sequences of the Illumina adapters and the 48 barcodes we are currently using.
Note that Bioo Sci. has recently started selling a set of 96 barcoded adapters. This set is not a simple expansion of the existing 48 barcodes. The 48 barcode set has 6 nucleotide barcode sequences in the adapter whereas the new 96 barcode set has 8 nucleotide barcode sequences.
I believe that Illumina is still selling just their original 12 TruSeq barcoded adapters. These adapters have 6 nucleotide barcode sequences. ***UPDATE, March 2012: Apparently Illumina are selling two sets of 12 barcoded adapters now for a total of 24. I don’t know the sequences of the second set but will add them below when I find out.
The barcodes of the 12 TruSeq adapters are identical to 12 of the Bioo Sci. barcodes, the first 12 but not in the same order, see below.
Here is the sequence information given below in an Excel doc.
Primers
| 5′->3′ | |
| Primer 1: | AAT GAT ACG GCG ACC ACC GAG ATC TAC AC |
| Primer 2: | CAA GCA GAA GAC GGC ATA CGA GAT |
Adapters
| 5′->3′ | |
| Universal: | AAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC T |
| Indexed: | GAT CGG AAG AGC ACA CGT CTG AAC TCC AGT CAC NNN NNN ATC TCG TAT GCC GTC TTC TGC TTG* |
* “NNN NNN” indicates the sequence of the 6bp barcode, see below.
Barcodes
| NEXTflex(Bioo Sci.) # | TruSeq(Illumina) # | Barcode5′->3′ |
| 1 | 2 | CGA TGT |
| 2 | 4 | TGA CCA |
| 3 | 5 | ACA GTG |
| 4 | 6 | GCC AAT |
| 5 | 7 | CAG ATC |
| 6 | 12 | CTT GTA |
| 7 | 1 | ATC ACG |
| 8 | 3 | TTA GGC |
| 9 | 8 | ACT TGA |
| 10 | 9 | GAT CAG |
| 11 | 10 | TAG CTT |
| 12 | 11 | GGC TAC |
| 13 | AGT CAA | |
| 14 | AGT TCC | |
| 15 | ATG TCA | |
| 16 | CCG TCC | |
| 17 | GTA GAG | |
| 18 | GTC CGC | |
| 19 | GTG AAA | |
| 20 | GTG GCC | |
| 21 | GTT TCG | |
| 22 | CGT ACG | |
| 23 | GAG TGG | |
| 24 | GGT AGC | |
| 25 | ACT GAT | |
| 26 | ATG AGC | |
| 27 | ATT CCT | |
| 28 | CAA AAG | |
| 29 | CAA CTA | |
| 30 | CAC CGG | |
| 31 | CAC GAT | |
| 32 | CAC TCA | |
| 33 | CAG GCG | |
| 34 | CAT GGC | |
| 35 | CAT TTT | |
| 36 | CCA ACA | |
| 37 | CGG AAT | |
| 38 | CTA GCT | |
| 39 | CTA TAC | |
| 40 | CTC AGA | |
| 41*** | GAC GAC | |
| 42 | TAA TCG | |
| 43 | TAC AGC | |
| 44 | TAT AAT | |
| 45 | TCA TTC | |
| 46 | TCC CGA | |
| 47 | TCG AAG | |
| 48 | TCG GCA |
***Bioo Sci. changed barcode 41 at some point. The alternative sequence is GCGCTA. As of March 2012 I’m not sure which one we have or which is the current one. If anybody has any trouble with barcode 41 check the alternative sequence – should probably be added to the de-multiplexing script as 41a and 41b.
Dan E.