We are doing 16S high throughput sequencing of endophytic bacteria. At the preliminary run we got few bacterial sequences and mostly plastid DNA.
Did anyone try to separate chloroplasts from bacterial genomes? Are there any suggestions for us as to how to increase the bacterial portion of the DNA?
Hi,
I hope that these article will be useful. Best Luck.
Article Endophytic bacterial community of a Mediterranean marine ang...
Article Practical innovations for high-throughput amplicon sequencing
Article A Method for Selectively Enriching Microbial DNA from Contam...
Article Development of a Bacterial Cell Enrichment Method and its Ap...
You can do this at the centrifugation stage, when the cells are first lysed to isolate and separate the chloroplasts from the bacteria. However, if you have passed this stage, you can also use complementary DNA/RNA oligos to specific chloroplast proteins encoded by the chloroplast DNA (e.g. P680/P700 reaction center proteins, etc) and have these oligonucleotides hybridised to agarose beads to trap the chloroplast DNA when performing a HPLC run, so that only the bacterial DNA will be eluted out.
Which hypervariable region in the 16S you are targeting? I did it last year considering V1-V2 and then later filtering with database. This worked perfectly in my case.
Please also check this discussion on RG to see whether it is useful for your project:
"How can I extract only bacterial (endophyte) DNA from plant tissue using kit?"
https://www.researchgate.net/post/How_can_I_extract_only_bacterial_endophyte_DNA_from_plant_tissue_using_kit
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