How to add illumina adaptors to amplicons?

[Neiltje]

How can you add Illumina adaptors to amplicons of +/- 300 bp? The ampliconlength is to small to use the nextera method. I tried by PCR by adding (a part of) the adaptor sequence to my specific primers but this failed. Then I tried to add them trough PCR already on my amplified sequence but this resulted in primer dimers. But there must be a way.

[nucacidhunter]

You may click this link for an Illumina published protocol. http://supportres.illumina.com/docum...15044223-b.pdf

[Neiltje]

well, this is exactly what I can't use for the reasons mentioned above

[nucacidhunter]

If this is not working for you, there are two other options. One is to ligate Illumina adapters post PCR to amplicons. The other one is to amplify flankig non- target region as well to obtain longer amplicons suitable for Nextera XT based approach.

In second approach that you have mentioned above, If your PCR results in desired amplicons you may be able to wash primer-dimers by cleaning up your PCR product with 0.8-1x Ampure beads.

[Neiltje]

Ligating adaptors sounds pretty good. I found a kit from New england biolabs with indexed primers. Problem is, these are primers, so single stranded? And my amplicon is double stranded. How do I get past this.

[nucacidhunter]

The easiest way would be to buy DNA library prep kit and skip end repair if you are using proof reading DNA polymerase and follow on by A tailing , adapter ligation and a few cycle of PCR.

[gwilkie]

The NEBnext kits have a double stranded multiplexing adapter in addition to indexed oligos - these kits are good value and work well for us.

Depending on your PCR enzyme you may or may not have to A-tail your amplicons before ligating the adapters. Then you can use indexed primers with a few cycles of PCR to add the index tags and resolve the forked ends. We use the NEBnext adapters/oligos with KAPA library prep kits, but there are lots of options out there.

Use 0.8x - 0.9x AMPureXP to purify your products and remove any primer/adapter dimers and go straight to sequencing.

If you are worried about low diversity during sequencing either pool different amplicons, sonicate before doing the adapter ligation or use extra PhiX spike-in. The new versions of RTA on the MiSeq are supposed to have solved this problem.

[Neiltje]

we are planning to use the NEBNext DNA library prep kit with the multiplexed oligo's. I need exactly 24 indices so I'm lucky :-).

Why are you using KAPA prep kit instead of NEB?

[gwilkie]

We use the KAPA Library Preparation Kit with KAPA Hifi hotstart - they are a good match for the NEBnext kits as KAPA do not supply oligos or adapters.

We use these on sonicated genomic DNA - so we need the end repair and A-tailing parts. You may only need some ligase for what you are doing, so may not be the best value for you?

The KAPA kits available in small sizes however, which are good if you do not have many preps. Half reactions work well also.

[Neiltje]

Well, my amplicons are blunt ended so I basically need the total kit. And I have +/- 90 preps. The only difference is the entry DNA, since they are amplicons, the concentration is a lot lower. On average around 200 ng, which is less than the recommended 1µg-5µg input. Should I alter the quantities of the reagents?

[gwilkie]

We are using picogram DNA inputs from clinical samples and our genome has high GC% and long repeat tracts making it challenging to amplify and sequence.

We found KAPA Hifi was the best enzyme for amplifying these - it has low bias and is very efficient. With your amplicons I'm sure any library prep kit will work fine.

IIRC the KAPA manual recommends 100ul reaction volumes. Library prep works fine for us using half and even quarter volumes (25ul). Saves a lot of cash

[Neiltje]

sounds great, do you use the recommended input concentration of adaptors? seems like overkill if your input DNA is so much lower.

[gwilkie]

Yes, use less adapters that's actually very important sorry I forgot to mention this!

I check my A-tailed libraries on the Qubit and use a 20:1 molar ratio of adapters. The idea is to maximise the efficiency of ligation and minimise formation of adapter dimers.

Most kits use adapter stocks at 15 micromolar. If you are not certain just reduce/dilute the adapter by the same ratio as your input DNA compared to recommended input DNA.

[Neiltje]

That's a clear answer, thanks :-).

[JeremyDay]

I agree that matching your adapter to input molarity should solve any problems with adapter dimers if you ligate. However, out of curiosity, have you tried doing the same with your primers? Primers are a lot cheaper than library prep with ligation and a quick titration of primers may help. I assume you'd be using primers with some truseq sequence then adding the rest in a second amp (flow cell and index)? I guess it is really all dependent on how much you are willing to spend on time or money. Sometimes it's worth fine-tuning if you have many experiments to do. I'm sort of surprised you'd need to lower you adapter conc if the input is amplicons, unless they are gel purified (maybe not clean amps?)