Ultra-low DNA yield library prep.

[francicco]

Dears all,

We're about to sequence a genome from a small insect. We can get ~500-1000 ng from 5 pooled individuals. But giving wild population should have high, maybe too high variability, we would like to build libraries from single individuals, maybe half of it, to avoid the gut, full of other stuff.

I was reading the Nextera XT DNA library preparation kit. They claim it is able to work with only 1ng of DNA.

Does anybody have some experience with it? Any other advice?

Thanks you a lot
F

[HESmith]

We routinely make libraries from 1-2ng for whole-genome sequencing/mutation identification in C. elegans (100Mbp genome) and obtain coverage comparable to bulk (1 ug) libraries. We've used a variety of library kits: Illumina ChIP, NEBNext Ultra DNA, Nugen Ovation SP+ Ultralow, and Diagenode MicroPlex.

[francicco]

Thanks!!!

What about ThruPLEX DNA-seq Kit?

F

[kerplunk412]

It sounds like you can get 100-200 ng of DNA per individual, and if that is the case I think you should consider PCR-free options. This will help with more even coverage of your genome and avoiding PCR artifacts and duplicates. The polymerases are pretty good these days, but nothing beats PCR-free. Also, PCR-free looks good to journal reviewers, if that is your eventual goal.

Also, unless there is a reason you need to go with Illumina, you may want to consider kits from other companies. You can often save money and get results that are as good as, if not better than, the comparable Illumina kit.

[francicco]

Thank you K.

So which kit do you would suggest? Or which company...

F

[nucacidhunter]

Nextera XT uses 1 ng input and it is robust for its recommended applications. The limitations are genome size (larger genomes will not get even coverage) and also GC content of genome. AT rich and GC rich genomes may fail or perform poorly.

I have not used ThruPLEX, but another product from this company performed well. They have good data comparing this product to competing ones and their protocol seems robust. If cost was not considered I would try this kit over Nextera. This protocol potentially can convert more of input DNA into sequencaeble library than Nextera.

[lac302]

Do you know the genome size and GC content of your insect genome?

I've yet to get optimal clustering out of multiplexed samples run through the Nextera XT bead based normalization, anywhere from 300-800k/mm2. I do routinely use the XT kit for single isolate libraries (120Mb genomes) that are run out on a fragment analyzer. If you go the Nextera XT route I would recommend doing a titration of input amount, e.g. 1, .8, .6ng.

[NextGenSeq]

I personally don't like Nextera since it shows insertion bias. Every Nextera library I've ever seen shows unusual GC content about 10 bp into the sequence read.

[francicco]

Hi guys,

the genomes I'm working with ranges from 300 to 500Mb.

It looks clear Nextera XT is not very suitable. So far ThruPLEX looks better. Do you guys agree?

thanks
F

[NextGenSeq]

I like the NEBNext Ultra kit or the NuGen Ultralow

https://www.neb.com/products/e7370-n...t-for-illumina

http://www.nugen.com/nugen/index.cfm...ry-systems-v2/

[lac302]

Are you planning on running these on a MiSeq?

[nucacidhunter]

Originally Posted by francicco

Hi guys,

the genomes I'm working with ranges from 300 to 500Mb.

It looks clear Nextera XT is not very suitable. So far ThruPLEX looks better. Do you guys agree?

thanks
F

According to supplier web data, this kit outperforms competing products and that looks logical for following reasons:

1- One tube format with no purification between library prep steps is expected to eliminate sample loss resulting in increased library diversity and reduced duplicates. Nextera is also one tube format, but at least ½ of input fragments will not be amplified or sequenced due to receiving the same adapter at both ends.

2- Adapter design and its addition to fragments eliminates adapter-dimer formation unlike NEB Ultra

3- Expected efficient adapter ligation because adapter to template ratio can be increased without formation of dimers

For low input applications such as this study where size of insect limits availability of DNA, it seems to be the right product. As you have noted assembly will be easier if DNA comes from one insect rather than multiple sample pool.

[NextGenSeq]

GC bias was more prominent in transposase-based protocols, particularly Nextera XT, likely through a combination of transposase insertion bias being coupled with a high number of PCR enrichment cycles.
Hum Immunol. 2015 Mar;76(2-3):166-75. doi: 10.1016/j.humimm.2014.

The NEBNext method uses Q5 polymerase which shows less GC bias

https://www.neb.com/products/m0541-n...pcr-master-mix

[francicco]

Actually I would like not to use PCRs...

[kobeho24]

Originally Posted by NextGenSeq

GC bias was more prominent in transposase-based protocols, particularly Nextera XT, likely through a combination of transposase insertion bias being coupled with a high number of PCR enrichment cycles.
Hum Immunol. 2015 Mar;76(2-3):166-75. doi: 10.1016/j.humimm.2014.

The NEBNext method uses Q5 polymerase which shows less GC bias

https://www.neb.com/products/m0541-n...pcr-master-mix

You mean use the Q5 polymerase after the tagmentation step of the Nextera XT procedure for amplification will reduce the GC bias causing by the tagmentation-based method?

[kerplunk412]

Originally Posted by francicco

Thank you K.

So which kit do you would suggest? Or which company...

F

Sorry I didn't respond to this earlier. The NEXTflex PCR-Free DNA-Seq kit from Bioo Scientific is what I would recommend, but please note that I work for Bioo Scientific. I believe Illumina and a couple of other companies offer similar kits. Feel free to PM me if you would like more information.

No matter which company you go with, I highly recommend PCR-free, using Covaris to shear your DNA. This eliminates concern of insertion bias and PCR bias, and I am fairly sure PCR-free is generally accepted to show the most even coverage over AT and GC rich regions. If you can get 100-200 ng of DNA from an individual I think PCR-free or very low cycle PCR library prep from an individual is feasible.

[SNPsaurus]

I would definitely be concerned with the presence of gut bacteria and endosymbionts. We do a lot of small insect genotyping by sequencing, and small insects have a higher probability of having lots of food DNA, gut bacteria, Wolbachia... you name it.

Can you get embryos from one female? A little heterozygosity would pale in comparison to having 2/3rds your reads go to species different from the one you are interested in assembling. Heads are another option. Samples sourced from heads have been very clean.