Custom multiplex sequencing barcodes / indexes (8-mer)

[JoeChris38]

Hi all,

I have synthesised a custom set of Illumina adaptors that use an 8-mer index to allow 384-plex multiplexing. I got these index sequences from the Nextera XT 384-plex index kit.

I am looking to expand my index set beyond 384-plex. Preferably beyond 1000-plex. Does anyone have the sequences for an index set, with good balance, that would allow me to expand my custom adaptor multiplex size up to 1000 (or beyond)? I would prefer if the indexes were 8-mers but could try longer indexes if this allow expansion of the set...

Thank you in advance for your help.

Joe

[MU Core]

Consider the 12 base index used in the following publication.

http://www.ncbi.nlm.nih.gov/pubmed/20534432

[Tom Barker]

There's a 16S amplicon paper (link below) with 32 i5 indices and 48 i7 indices which shoud be enough for 1536 libraries. The index sequences are in the supplementary material. The indices are dual 8bp similar to Nextera XT

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3753973/

[JBKri]

I suppose this is the two-step PCR approach? If so you could also add a short secondary index between the specific primer and the tail, to make it "double-dual" indexing. With two pairs of tailed, specific primers; one normal pair, and one pair with the secondary index inserted, you quadruple the number of possible combinations. In addition, the secondary index can also act as a heterogeneity spacer, thus helping with base diversity. Some manual demultiplexing will be required. We have done this a couple of times, it worked well.
Jon