I am working on the microbiome of plant seeds. I am planning to send extracted DNA from the seeds for shotgun sequencing. But the extracted DNA will be -for sure- full of plant DNA. Since shotgun sequencing is quite expensive, I don't want to waiste money on sequencing the plant genome. So to make it more effective, I am looking for a protocol or kit that can deplete the host DNA.
I would be very greatful if someone could help me with this problem.
Plant host DNA and RNA can significantly interfere with microbial community analysis in seed microbiome studies. The most effective protocol involves a multi-step approach combining physical and enzymatic treatments. First, seeds should be surface-sterilized using a sodium hypochlorite solution followed by thorough rinsing with sterile water. The seeds are then mechanically disrupted through grinding or bead-beating in a buffer containing proteinase K and DNase I. This step breaks down the seed tissue while beginning to degrade host nucleic acids. A differential centrifugation step follows to separate microbial cells from plant debris. The microbial pellet is then treated with a DNase/RNase cocktail specifically optimized to target plant host nucleic acids while preserving microbial genomic material. Some protocols also incorporate size-based filtration steps to further remove plant debris. Throughout the process, it's crucial to maintain cold temperatures and use RNase-free reagents to preserve microbial nucleic acid integrity. The effectiveness of host depletion should be validated using qPCR targeting plant-specific genes before proceeding with downstream microbiome analysis.
Thank you very much for your answer. I would like to try this method. So, do you have a detailed protocol or can you recommend a paper that used this method?
- Badges
- Science method