Range of DNA (ng) Necessary for Illumina Sequencing

[roliwilhelm]

Hello,

I'm attempting SIP-metagenomics and the limiting factor is the quantity of DNA I will recover for sequencing. So far I'm able to recover ~30ng of enriched DNA (which is not bad!), but estimates for paired-end, Illumina Hi-Seq say minimum 500ng. Now, I'm caught balancing between whether to do whole genome amplification or pool multiple SIP-DNA runs to get as close to 500ng as possible. The latter is costly in time and reagent, but would likely produce a less biased sequence library. I am wondering whether anyone has advice on that, AND, more importantly, what the lower limits for quantity of DNA are for Illumina library prep?

Thanks in advance,

Roli

[HESmith]

ChIP-Seq libraries are constructed from 1-10ng input DNA. You could adapt the protocol for your application.

[roliwilhelm]

Thanks for the input. I looked up ChIP-Seq and found that it employs special sequencing methodology (cluster sequencing) which seems like an extra amplification step of bound DNA. The whole ChIP part of the process isn't useful in my case b/c I'm looking at total DNA (not specific components bound or bindable to protein). Still, you're comment is helpful b/c it suggests there is alternative technology to MDA.

[HESmith]

All Illumina sequencing uses cluster generation, so your objection is unclear. Perhaps it would be useful for you to review the technology. And you misunderstood my reference to ChIP-Seq; the salient point is that it's feasible to construct libraries from small amounts of input DNA without amplification.

[roliwilhelm]

Yep! Thanks for the info! I did get your point and, as anyone new to sequencing technology knows, it can be difficult to get to heart of the subject b/c every answer is tempered towards a specific application and every webpage (at least @ Illumina) is selling a different kit and their selling points don't generalize easily.

[snorberg]

Hey,

If you are using the TruSeq library prep kit, than yes, 500ng of DNA is the recommended input. However, if you use Illumina's Nextera or Nextera XT library prep kits, the required input is 50ng and 1ng respectively. The protocols are also much quicker and easier.

Nextera Product Info: http://www.illumina.com/products/nex..._prep_kit.ilmn

Hope that helps.

Best, Steve

[roliwilhelm]

Great! Thanks Steve!