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jov14 04-24-2018 09:30 AM

Creating Custom dual index primers for Illumina Seqeuncing
I need help (or tips) for creating my own indexing-primers for Illumina seqeuncing (TrueSeq-compatible):

It seems to me that I could simply devise my own index primers based on the Trueseq adapter sequences and currently available index primer sequences, by simply exchanging the 8bp index with whatever I want. Then I could have these primers synthesized by e.g. Eurofins (or whatever), and use them instead of the indexing kits sold by Illumina and/or NEB, right?

Does anybody have experience in this? Are there any specific pitfalls to this, I should look out for? Do the Index-primers have to be modified in any way?


My lab is doing bacterial single cell and transcriptome sequencing, both of which produce uneven sequencing coverage.

Currently we are switching from sequencing on MiSeq & Nextseq-plattforms to using the HiSeq Xten, since we get much more output for the money (and have extremely good offers for that). With the number of libraries we need currently to sequence, using the older Illumina plattforms is definitively not feasible for us anymore.

However this Illumina plattform (and all subsequent new illumina plattforms as well) has a massive problem with "adapter hopping", due to the relatively new "ExAmp" cluster amplification method. This problem is even worse, to the point of being intolerable, when having uneven sequence coverage, such as transcriptomes or MDA'ed Single Cell sequencing libraries.

However, it seems this problem can be largely contained simply by using unique barcodes at each fragment end (currently in most dual indexed libraries there are only unique combinations, each reusing either the forward or the reverse index)

Sadly, the commercially available dual indexing kits for illumina libraries each offer only 8 forward and 12 reverse indices, allowing only for 8 double-unique indexed libraries (or up to 16 if you combine both of the available kits). This is not an option for us, as we have to multiplex at least 70 samples.

I do not understand this index limitation, as in theory it should be possible to create 4^8 = 65536 unique 8mer indices, so even when removing all extremely low-complexity options there should still be more than enough distinct 8mer-sequences left to create 192 unique indices (giving me 96 unique forward and 96 different unique reverse indices), right?

Even though this problem is receiving more and more attention these days, it seems no such option is commercially available yet. Therefore I need to create my own index-primers and want to make sure I take all necessary considerations into account.

nucacidhunter 04-25-2018 05:09 AM

There are commercially available unique duel indices from venders such as Illumina, NEB, Bioo Scientific, NuGEN (up to 96 UDI) and Integrated DNA technologies (up to 384). These are offered as adapters (except NEB) and you could use them to design unique duel index primer set for your application. The advantage of using these is that they have been validated and less likely to cause any issue.

TruSeq kits based on ligating duel indexed adapters (either combinatorial or unique) do not use indexed primers.

jov14 04-25-2018 05:16 AM

Thanks, I missed those options somehow, when I was looking for them earlier...
Converning the use of primers with TruSeq kits, We are actually mostly using NEB library prep kits which are Trueseq compatible, but do use indexed primers. So I would have had to create alternative primers based on Truseq adapters, even though I would not have been actually using Illumina TruSeq-kits.
But I strongly agree on preferring validated commercially available kits over home-made solutions.

nucacidhunter 04-25-2018 05:46 AM

NEB recently released unique duel index primers and it should be easier to use them with NEB Illumina libray prep kit.

pmiguel 04-25-2018 09:48 AM


Originally Posted by nucacidhunter (Post 216826)
TruSeq kits based on ligating duel indexed adapters (either combinatorial or unique) do not use indexed primers.

Although, a little weirdly, the Epicentre "scriptseq" kit is sold by Illumina and that uses step-out PCR to add the indexes, etc.

Well, not indexes but "index"-- I don't think ScriptSeq uses dual indexing natively. But because the final products are TruSeq-identical it is trivial to design UDI's for them.

Also, it looks like Swift Bioscience has a UDI kit as well?


SNPsaurus 04-25-2018 03:24 PM

You should check out the Illumina free adapter blocking kit that they recently released to mitigate the examp index hopping. You might still want to go dual unique, but just keep it in mind.

NGSMicro 04-26-2018 06:40 AM

I have been looking at ordering my own custom indices - I have hypothetically made some like this:


Where the first section binds to the flow cell, the lowercase characters in the middle are my custom index and the final section is my sequence adapter. I haven't used these yet if anyone knows whether they will work as it comes in about 30% cheaper than IDT's UD indices.

If you want help generating the barcodes themselves I found this useful: http://comailab.genomecenter.ucdavis...code_generator

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