As any of you that used our two-enzyme GBS protocols know, the current GBS adapters are not set up for index reads – they were designed before Illumina introduced indexing. That is an issue when we want to pool those libraries with other indexed Illumina libraries; we always have to spend a bunch of time explaining the situation to the sequencing centres, and often end up having to do the demultiplexing ourselves.

No more! I have modified the adapters and primer designed so that you can get indexed GBS libraries, with adapters that are identical to standard TruSeq Illumina adapters (so they will work on any Illumina sequencer). The current “barcoded” adapters (PE1, i5) are actually fine, and you just need to use the indexed i5 primers we use for WGS libraries instead of the standard GBS PCR F primer. I had to re-design the Y-shaped “common” adapters (PE2, i7), as well as the corresponding primers. A list of all adapters, primers, in-line barcodes and indices is attached to the post.

Besides not having to worry about compatibility with other libraries when sequencing GBS libraries, adding indexes has of course the advantage of increasing the potential to multiplex GBS samples in a same lane. I kept the original 192 barcodes on the barcoded adapters and 12 barcodes on the common adapter; we already had 48 compatible indexed i5 primers, and I designed 24 indexed i7 primers – so you could potentially pool 192 * 12 * 48 *24 = 2,654,208 samples in a single lane. That’s probably not going to happen, but you can use the indices as an additional check to distinguish your libraries.

The barcoded adapters are still in Loren’s lab, but the rest of the adapters/primers are with me – just come and get them if you need them (my lab is in MSL385), or if you want to prepare your own aliquots.