Sequencing 16S rRNA gene amplicons is the gold standard to uncover the composition of prokaryotic communities. The presence of multiple copies of this gene makes the community abundance data distorted and gene copy normalization (GCN) necessary for correction. Even though GCN of 16S data provided a picture closer to the metagenome before, it should also be compared with communities of known composition due to the fact that library preparation is prone to methodological biases. Here, we process 16S rRNA gene amplicon data from eleven simple mock communities with DADA2 and estimate the impact of GCN. In all cases, the mock community composition derived from the 16S sequencing differs from those expected, and GCN fails to improve the classification for most of the analysed communities. Our approach provides empirical evidence that GCN does not improve the 16S target sequencing analyses in real scenarios. We therefore question the use of GCN for metataxonomic surveys until a more comprehensive catalogue of copy numbers becomes available.

• Klíčová slova
• 16S rRNA, Gene, Metataxonomic surveys,
• MeSH
• genová dávka MeSH
• genová knihovna MeSH
• metagenom genetika   MeSH
• metagenomika normy   MeSH
• mikrobiota genetika   MeSH
• RNA ribozomální 16S genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• RNA ribozomální 16S MeSH

Biomolecules for OMIC analysis of microbial communities are commonly extracted by bead-beating or ultra-sonication, but both showed varying yields. In addition to that, different disruption pressures are necessary to lyse bacteria and fungi. However, the disruption efficiency and yields comparing bead-beating and ultra-sonication of different biological material have not yet been demonstrated. Here, we show that ultra-sonication in a bath transfers three times more energy than bead-beating over 10 min. TEM imaging revealed intact gram-positive bacterial and fungal cells whereas the gram-negative bacterial cells were destroyed beyond recognition after 10 min of ultra-sonication. DNA extraction using 10 min of bead-beating revealed higher yields for fungi but the extraction efficiency was at least three-fold lower considering its larger genome. By our critical viewpoint, we encourage the review of the commonly used extraction techniques as we provide evidence for a potential underrepresentation of resistant microbes, particularly fungi, in ecological studies.

• MeSH
• Bacteria genetika   MeSH
• bakteriální léková rezistence genetika   MeSH
• bakteriální proteiny chemie   izolace a purifikace   MeSH
• DNA bakterií genetika   MeSH
• DNA chemie   izolace a purifikace   MeSH
• fungální léková rezistence genetika   MeSH
• fungální proteiny chemie   izolace a purifikace   MeSH
• houby genetika   MeSH
• mikrosféry MeSH
• vibrace ultrazvukové metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• bakteriální proteiny MeSH
• DNA bakterií MeSH
• DNA MeSH
• fungální proteiny MeSH