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memento 09-26-2012 07:33 AM

Solutions for multiplexing/barcoding
Dear All,
Can somebody point me out to a good and reasonably priced barcoding solution for illumina's MiSeq ? Are the adaptor sequences similar between MiSeq/HiSeq ? I found a barcoding generator by comai, but there is no protocol how to use it's output in a lab setting (probably very trivial question). I would assume that this is the cheapest solution. Bioo has a 96samp kit good for 768 reactions (question is how big rx they are talking about?) for 4995$. Are they the cheapest commercial solution ?
Thanks in advance!

TonyBrooks 10-15-2012 05:00 AM

What application are you specifically looking at? RNA-Seq, DNA-Seq, ChIP-Seq, other...
What level are you looking to plex (12, 48, 96, higher?)
IMHO indexing primers are the most over-priced aspect of NGS. If you think you are going to be doing a lot of index library preparation, you may want to look at making your own.

We ordered a set of indexes based on the above paper and use them regularly for both indexed (and non-indexed runs - you don't need to read the barcode). It works to be significantly cheaper per reaction than buying commercially, but there is obviously a largish initial outlay on the HPLC oligos. (Of course, you don't need to order all 96 in one go - one synthesis should provide enough for hundreds of reactions).
Those oligo sequences work well on the GAIIx, HiSeq and MiSeq - just remember to use the custom index sequencing primer.

There are also people on here dabbling with homebrew dual indexing, which is something we may look at in the future. It cuts the number of oligos you need to order down and will hopefully be scalable to >96 plex.

JackieBadger 10-15-2012 05:23 AM

for MiSeq amplicon work we use the 454 MIDs

bhennuy 12-05-2012 06:13 AM

indexes sequence for multiplexed > 96
We are interested in using dual indexing strategy for multiplexing more than 384 samples in one run on MiSeq instrument. Could we use the 48 indexes (6 bases) provided by Illumina for Truseq Small RNA sample prep as i7 indexes ? how to add 2 bases to these sequences to get unique 48 indexes ? We would use 8 i5 (as Nextera) indexes to reach 384 unique combinations. Do you think this strategy would work? Could we use more than 8 i5 indexes ?
Thans for your help.

TonyBrooks 12-05-2012 06:25 AM

You could look at this paper from Sanger
They use 96-plex 8-mer barcodes. You could use those sequences for your i7 indexes.

bhennuy 12-05-2012 08:43 AM

Thank you TonyBrooks for the info, I will read it carefully. Do you know why Illumina do not provide more i5 indexes to increase the number of multiplexes ?

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