I have environmental DNA samples extracted using the MoBio/Qiagen PowerWater kit and quantified using a Quantus fluorometer. While most of my samples have concentrations of >100ng/uL dsDNA, a few have around 15-30ng/uL (in 100uL total volume) which might not be able to satisfy the 2,000ng input DNA required for PCR-free sequencing.
Will it still be possible to submit these samples for PCR-free shotgun metagenomic sequencing (150bp paired-end)? Or would my only option be to re-do sampling with more biomass for extraction?
*I really would not prefer to do PCR-based sequencing or metagenomics because of potential bias introduced by the technique.
What volume of starting material did you use? I'd assume that if you submit less than 2,000ng, then your sequencing depth will be affected as there is less material to sequence. How easy would it be to collect more starting material?
Hi Luke! We collected 2.0L volumes of water and though I am aware of the variety of volumes in published literature (I came across a study where they pumped 500.0L of well water to a filter), my samples were quite turbid so I was confident that they would all yield comparable and enough amounts of DNA.
We collected quite a distant place (active mines) but if all else fails then I guess a sampling trip would be the best recourse for us.
So you are sequencing all extracted material, and not enriching for any marker(s) in particular?
For the samples at 15-30ng/ul in 100 ul, assuming quality is there, I would think these would be fine after concentrating down to a smaller volume. Most facilities that I've seen require 1-2 ug for their PCR-free preps. Are you doing the preps in-house, or at a facility?
Could it be an option to use PCR during library preparation, but then collapse PCR-duplicated polymorphisms by identifying them bioinformatically, either using length polymorphism, or by incorporating a degenerative region into your adaptor design, thereby mitigating (or at least minimizing) any bias? The latter would only be possible if you are doing the preps in-house, and if custom adaptors is an option, but detection using contig lengths could still work.
You could also add some amplicon dna that you’re fairly certain won’t be in your metagenome to bulk out your sample. Have seen papers do this with 16S when theyre after viral dna. Disadvantage of this approach is that it does reduce coverage. Could you also barcode your samples allowing you to multiplex them?
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