CeGaT Researches on Best Practice for Microbiome Analysis

CeGaT addresses the urgent need for standardized microbiome analysis workflows by comparing three commonly used sequencing library protocols.

So far, a scientific “best practice” consensus for shotgun metagenomics has not been found. Data quality, analysis accuracy, and repeatability of studies strongly vary, depending on the wet lab protocol and analysis workflow chosen by the scientist. Standardized, highly accurate microbiome analysis workflows could contribute to study comparability. For providing more standardization, CeGaT tested key quality parameters of three commonly used sequencing library protocols for shotgun metagenomics:

•             Protocol A: Illumina Nextera DNA Flex (input: 100 ng)

•             Protocol B: Illumina Nextera XT DNA (input: 1 ng)

•             Protocol C: Down-scaled Illumina Nextera XT DNA (input: 0.1 ng).

The different libraries were sequenced using the same parameters. The generated data was mapped against a database. The analysis focused on the correct representation of a microbial community’s composition, detectable diversity, and repeatability.


To test the performance of the protocols, we compared the three library preparations using the same standard. We used a commercially available microbial community standard that consists of DNA from ten microbial species at a known composition.

All protocols were able to capture the microbial species present in the standard. However, regarding the correct representation of the community composition (i.e. the theoretical relative sequence abundance of the detected microbial species) the protocols revealed differences. The theoretical composition was most accurately determined with protocol A.

Detectable microbial diversity and repeatability

For the investigation of repeatability and detectable diversity, we used a complex microbial DNA pool, generated from ten individual stool samples. We performed three library preparations for each protocol using this complex microbial DNA pool.

All protocols were able to detect the most common gut microbiome representatives and covered a wide range of different microbes. To evaluate the microbial diversity within a sample, we calculated the species richness and evenness as well as the Shannon Diversity Index. Each parameter was slightly higher with protocol A.

The highest repeatability was also shown by Protocol A, though the difference was slight. In comparison with protocol B and C, protocol A had the lowest variation between replicates among the most abundant taxonomic units.


Our detailed comparison of three different library preparation protocols for shotgun metagenomics revealed that protocol A achieved the best results for both the microbial community standard and the complex stool samples. Based on these results, protocol A represents an accurate standard protocol and is highly suitable for metagenomic profiling. Furthermore, protocol C, which showed only a slightly lower accuracy than protocol A, seems to be a good alternative for extremely low concentrated DNA samples due to its very low input-requirements.

Further information on the study and the results can be found in our Tech Note.