NEB multiplex adapter kit

[murpheli]

Hi I know the discussion about Illumina Barcode/adapter sequences has been beaten to death on here, but I'm curious if anyone is currently ordering the multiplex adapter kit from NEB and using it in conjunction with say a KAPA library prep kit? I hear the KAPA enzyme is superior to most, but they don't offer the indexing/adapters. Trying to piecemeal a protocol since Illumina is discontinuing the Truseq library prep. I know I can order these oligos through IDT, but I cannot seem to get straight answers from them fast enough right now.

[ECO]

I use the adapters from Bioo and am pleased with the results.

I haven't used those from NEB, my only concern is that they use a hairpin containing a uracil which requires an additional enzymatic treatment with USER (UDG+Endo) to break the hairpin (and in their opinion, getting around Illumina Y adapter IP).

Kapa has a document that specifies what to order from IDT...if you can't find it let me know and I'll try to dig it up.

[microgirl123]

We also use the BioO adapters and have been happy with them. They also sell library prep kits.

[FroggyFlox]

I've also been using the adapters/barcodes from BIOO, they work nicely and are also compatible with the KAPA quantification kit if this is your concern. My understanding is that any adapter compatible with the Illumina platform would also be compatible with the KAPA qPCR quantification kit, but I would be glad if someone could prove me wrong on this.

Regarding the NEB adapters, I actually just ordered them and will give them a try. A colleague from my institution tried them and was happy with them, happier than with Illumina's. He had better results regarding primer-dimers.

Looking forward to hear any more feedback on the NEB adapters though...

[henry.wood]

We switched to NEB library preps a few months ago and are very pleased with them. They can produce good, libraries with no adapter peaks from samples which had previously failed with truseq due to poor quality/low amounts of DNA.
We also custom designed an extra 200 tags based on the NEB primer sequence and got them from IDT to increase our tagging options. Works a treat.

[ijivanjee]

Hi - I’m the product manager for next gen sequencing applications at IDT. Just wanted to respond here and apologize to murpheli in regards to her experience. The difficulty we face at IDT is that, as a manufacturer of custom oligos, there really are a lot options we provide. I recognize that this could be overwhelming.

I know there are many out there that have purchased adapters from us with great success and I would love to hear your opinions on what things were important to you.

Again, I sincerely apologize for your frustration. I am glad to be aware of this issue and I will work to make the process easier in the future.

[Christopher Odom]

Originally Posted by FroggyFlox

My understanding is that any adapter compatible with the Illumina platform would also be compatible with the KAPA qPCR quantification kit, but I would be glad if someone could prove me wrong on this.

Dear FroggyFlox,

The KAPA Library Quantification Kit- Illumina can be used to quantify any library made with a full-length Illumina adapter after ligation and/or after library amplification. The qPCR quantification primers used in our kit are based on the Illumina flow cell primers (P5 and P7 sequences). In other words, if the library will cluster on an Illumina flow cell, it can be quantified with the KAPA Library Quantification Kit- Illumina.

I hope this is helpful! If you have any additional questions, please feel free to contact Kapa Biosystems Scientific Support at support@kapabiosystems.com. Thank you!

Christopher Odom
Technical Applications Scientist
Kapa Biosystems

[gwilkie]

I've been using the NEB adapters for a year or so and am very happy with them. They work great with the Kapa library prep kits and Kapa qPCR quantification. The index sequences are the same as TruSeq LT.

I have no problems with adapter dimers - but I usually measure the amount of A-tailed DNA and add the adapter in a 20:1 molar ratio.

Cheers, Gavin

[kbushley]

Hi Gavin,

I am also wanting to try to adjust adaptor concentration but I can't find anywhere the actual concentration of the NEB Adaptors (cat. #E7337A). Do you have that info?

[gwilkie]

Hiya, yes the concentration of the NEB adaptor is 15 uM (micromolar).

It only tells you this in some of the NEB manuals (e.g. NEB #E7335S) but I would imagine the adaptor is the same concentration in all their kits. Certainly this works very consistently for me.

When you use less adaptor, it is also possible to use less USER enzyme and do smaller reactions for end repair, A-tailing, ligation etc.

The whole library synthesis scales down very well, and works for me with tiny amounts of input DNA. However I am sequencing viral genomes which are relatively small... so we don't need much DNA to cover the genome many times.

Originally Posted by kbushley

Hi Gavin,

I am also wanting to try to adjust adaptor concentration but I can't find anywhere the actual concentration of the NEB Adaptors (cat. #E7337A). Do you have that info?