Questions about NextraXT sample prep

[barak yaacov]

Dear Friends
My name is Dr Barak Yaacov, and I am working in a medical center, we bout a Miseq to our lab and I am trying to deep sequence several genes together about 80 amplicons as a first pilot.

I decided to begin with Nextera XT kit, I design the amplicon to size between 400-900 bp. Run each one of them on gel separately clean on colons, polled together, majored by Qubit , and viewed on a bioanalyzer.
I started library prep using the NexteraXT kit fuelled all protocol lode the sample on a cartage and the run failed. At first I thought I had a problem with the normalization step. The scant time I stop after the PCR cleanup stage lode on bioanalyzer and got nothing I lost all amplicon in the way. Could it be that I need to use x1.8 AMPure XP (90ul) instead of x0.6 AMPure XP (30ul)?
When I look at the protocol of AMPure XP they recommend using x1.8 AMPure XP.
Moreover is it possible to skipped the normalization stage and after tagmentation ,PCR and clean up run the samples on bioanalyzer polled them together as in the TruSeq protocol? Since post normalization step you can't run your sample on bioanalyzer (single strand DNA) you loss an important QC step before starting the run. How much Phix should I add my sample after using the Nextera XT kit?
Many Thanks,
Barak

[TonyBrooks]

Why are you using Nextera if your amplicon size is 400-900? The Nextera kit will shear your DNA in addition to adding in the Illumina adapters. As your DNA is short to begin with, it's probably completely chewed up your amplicons - hence the failed run. Did you run the Bioanalyser on on the library before loading onto the MiSeq. You probably wouldn't have seen a product.
The varying Ampure volumes will size select at different points. 1.8X is the generic volume and will remove anything below 100bp. The less you put in, the higher the size exclusion, so 0.6X will remove most things below about 250bp (which would be your Nextera chewed DNA).
Redesign the PCRs to be longer (LRPCRs >5kb) to cover your genes, then Nextera prep.

[pmiguel]

Although the LRPCR approach would be ideal, in many cases it isn't realistic. NexteraXT should be able to deal with amplicons in the 400-900 bp range okay.

But I think you are right, the 0.6x AMPure will remove most of the products you want. If possible, do a high sensitivity chip after AMPure to see what is there. Subtract 130bp from the sizes you see -- that will be your insert sizes. If acceptable, you should be fine. Whether you use normalization or not.

If you don't normalize, you will need to use the NaOH denaturation procedure, rather than the heat denaturation (which is for ssDNA and may not work at all for dsDNA).

BTW, even if you do normalize, qPCR is still an option for QC.

--
Phillip

[TonyBrooks]

I can't see how Nextera would work on amplicons of that size. You will need at least two insertion events in each amplicon to be able to generate library (as you need the adapters on both 3' and 5' ends). Plus you will lose around 50bp of the terminal ends due to non-transposition in those regions. I just think it's highly unlikely you could get anything at the end.

[pmiguel]

I can't see how Nextera would work on amplicons of that size. You will need at least two insertion events in each amplicon to be able to generate library (as you need the adapters on both 3' and 5' ends).

I am with you -- I find it surprising as well. But it does seem to work. Or at least I have seen it work for amplicons even smaller than 400 bp.

Plus you will lose around 50bp of the terminal ends due to non-transposition in those regions. I just think it's highly unlikely you could get anything at the end.

Yes, I have hear this. So adding 50 bp on each end, if possible, is warranted.

--
Phillip

[barak yaacov]

Thank you for all for your responses
There are two Nextera kits: The Nextera XT should work on amplicons up to 1000bp while the Nextera kit work on amplicons up to 5000bp.
I did the library prep today again and after PCR clean up I run the sample on bioanalyzer and thy look good pick about ~450 bp so the 0.6x AMPure work. I think I will skip the normalization step of the protocol and do it manually. What is the final concentration I can upload on the cartage of the miseq? If I have difference between the concentrations majored on Qubit and the one from the bioanalyzer to which one should I regard?
Many thanks
Barak

[stinky]

Pmiguels you are right, I took this off of the Nextera XT page at illumina....

Smaller amplicon inputs into Nextera XT preps typically yield smaller insert size ranges. To maximize recovery of smaller fragments out of the AMPureXP cleanup we recommend > 300 bp to ensure even coverage across the length of the DNA fragment. An expected drop off in sequencing coverage about 50 bp from each distal end of a fragment may be seen. This is because the tagmentation reaction cannot add an adapter right at the distal end of a fragment. For PCR amplicon sequencing this can be easily averted by simply designing your amplicons to be ~100 bases larger than the desired insert to be sequenced.

Are there any protocol differences based on different amplicon lengths?
For amplicons greater than 500bp, Illumina recommends using a 0.6x AMPure XP cleanup (30 μl volume of beads). For amplicons less than 500bp, Illumina recommends using a 1.8x AMPure XP cleanup (90 μl volume of beads) to maximize yield."

[JBKri]

Pmiguels you are right, I took this off of the Nextera XT page at illumina....

Smaller amplicon inputs into Nextera XT preps typically yield smaller insert size ranges. To maximize recovery of smaller fragments out of the AMPureXP cleanup we recommend > 300 bp to ensure even coverage across the length of the DNA fragment. An expected drop off in sequencing coverage about 50 bp from each distal end of a fragment may be seen. This is because the tagmentation reaction cannot add an adapter right at the distal end of a fragment. For PCR amplicon sequencing this can be easily averted by simply designing your amplicons to be ~100 bases larger than the desired insert to be sequenced.

Are there any protocol differences based on different amplicon lengths?
For amplicons greater than 500bp, Illumina recommends using a 0.6x AMPure XP cleanup (30 μl volume of beads). For amplicons less than 500bp, Illumina recommends using a 1.8x AMPure XP cleanup (90 μl volume of beads) to maximize yield."

Reviving an old thread here....
We have PCR-products without Illumina adapters (~500 bp), and we are thinking to use the Nextera XT kit to add the adapters and indexes.
Am I correct in assuming the resulting reads will be a mix of various lengths (up to 400 bp) and different parts of the insert? Can such data be used for e.g. Qiime processing?