Hi Barryette Oberholzer, the DNA smear you mentioned, starting at the 10,000bp marker and extending down to the 2,000bp marker or slightly lower, is within the expected range for soil DNA extracted using the DNeasy PowerSoil Pro Kit. This smear indicates a range of DNA fragment sizes, which is common in environmental samples like soil.

For metabarcoding using 16S and ITS primers, this DNA fragmentation should generally not have a considerable effect on the relative abundances of the microbial communities you are studying. Metabarcoding targets specific regions of the 16S ribosomal RNA gene (for bacteria/archaea) or ITS regions (for fungi) within the DNA samples. The primers used for metabarcoding typically amplify relatively short DNA fragments within these regions (e.g., around 200-500 base pairs). Even though your DNA smear extends beyond this typical range, it is unlikely to impact the relative abundances significantly. This is because during PCR amplification with the metabarcoding primers, the shorter target fragments within the range of interest will be preferentially amplified. The longer DNA fragments in the smear are less likely to be amplified efficiently.

However, it's important to note that the precise impact of DNA fragmentation on relative abundances can vary depending on the specific primers used, the target region being amplified, and the characteristics of the environmental samples. It's always a good idea to validate the primer efficiency and assess the metabarcoding results using appropriate positive and negative controls to ensure accurate interpretation.

Hi John Harvey Santos

I'm planning to conduct Metagenomic analyses on DNA extracted from soil using the ITS and 16S primers. However, I'm getting bands at 2k or 3k on the gel, even though my ratios and DNA concentration are within the acceptable range. I was expecting a thicker band above 10k. I'm concerned about the quality. Can I proceed with the metagenomic analyses or should I extract the DNA again modifying my method? Do I have too much smears? I use Dneasy Pro kit for soil extraction.

Hi Fernanda Pereira, it's important to ensure the quality of your DNA samples for accurate metagenomic analyses. The presence of bands at 2k or 3k on the gel, instead of a thicker band above 10k, may indicate potential issues with your DNA extraction or PCR amplification. It's possible that you have smears, which can affect the quality of your results. Some suggestions that might work for you:

• Check the integrity of your DNA: Run your DNA samples on an agarose gel without PCR amplification to verify if the smears are present before amplification. This will help determine if the issue lies with the DNA extraction or subsequent steps.
• Optimize your DNA extraction protocol: Ensure that you're following the manufacturer's instructions PRECISELY for the Dneasy Pro kit. Consider modifying your extraction protocol, such as adjusting the lysis conditions or using alternative kits, to improve DNA quality and yield.
• Optimize PCR conditions: Review your PCR conditions, including annealing temperature and primer concentrations, to ensure optimal amplification of the target regions. Adjusting these parameters might help obtain the desired band size.
• Consider re-extracting DNA: If you consistently observe smears or unsatisfactory results, it may be worthwhile to repeat the DNA extraction process using an alternative method or modified protocol. This could help improve the quality of your DNA samples for more reliable metagenomic analyses.

Thanks a lot for the detailed answer John Harvey Santos!