Comparison of Three DNA Isolation Methods and Two Sequencing Techniques for the Study of the Human Microbiota

Breast cancer is the most commonly diagnosed cancer in women and the second leading cause of female death. Altered interactions between the host and the gut microbiota appear to play an influential role in carcinogenesis. Several studies have shown different signatures of the gut microbiota in patients with breast cancer compared to healthy women. Currently, there is disagreement regarding the different DNA isolation and sequencing methodologies for studies on the human microbiota, given that they can influence the interpretation of the results obtained. The goal of this work was to compare (1) three different DNA extraction strategies to minimize the impact of human DNA, and (2) two sequencing strategies (16S rRNA and shotgun) to identify discrepancies in microbiome results. We made use of breast tissue and fecal samples from both healthy women and breast cancer patients who participated in the MICROMA study (reference NCT03885648). DNA was isolated by means of mechanical lysis, trypsin, or saponin. The amount of eukaryotic DNA isolated using the trypsin and saponin methods was lower compared to the mechanical lysis method (mechanical lysis, 89.11 ± 2.32%; trypsin method, 82.63 ± 1.23%; saponin method, 80.53 ± 4.09%). In samples with a predominance of prokaryotic cells, such as feces, 16S rRNA sequencing was the most advantageous approach. For other tissues, which are expected to have a more complex microbial composition, the need for an in-depth evaluation of the multifactorial interaction between the various components of the microbiota makes shotgun sequencing the most appropriate method. As for the three extraction methods evaluated, when sequencing samples other than stool, the trypsin method is the most convenient. For fecal samples, where contamination by host DNA is low, no prior treatment is necessary.