Challenges of Next-Generation Sequencing targeting Anaerobes.
Conrads G, Abdelbary MMH
Anaerobe. Feb 2019
COMMENT: Next generation sequencing is currently the method of choice for analyzing complex microbial communities. As a culture-independent method, it is specially useful for the study of anaerobes and anaerobic infections. However, results obtained could be greatly influenced by technical factors. In this review, the authors focusing on some of these variables.
To obtain appropiate NGS results, the authors highlight some recommendations:
- A contamination-free collection of the sample.
- If a direct processing of the fresh sample is not possible, rapid freezing to -80ºC is the best option. Repeated freeze-thawing cycles should be avoided.
- An efficient DNA extraction protocol (including bead beating and a lysozyme - mutanolysin buffer) is essential to obtain a good bacterial community structure representation.
About sequencing machines:
MiSeq have longer reads and lower error rates compared to PGM, which in return had the fastest turn-around-time.
For 16S - based amplicon sequencing:
As a perfect methodology does not exist, laboratories interested in specific (e.g. anaerobic) microbes should optimize their protocols to accurately detect the key taxa of central interest together with the widest range of accompanying species.
The Silva-TestPrime online tool helps to find the optimal 16S directed primers for individual studies.
For metagenome profiling, the authors give some recommendations to select the best classifier tool.
In conclusion of this review, an optimized sampling and DNA extraction method together with 16S long read amplicon sequencing or shotgun sequencing is a conditio sine qua non for a complete microbiome analysis in mixed anaerobic infections and environments. During sampling, the overall safety instructions have to be expanded shielding the specimen from environment to avoid contaminants. We also recommend that laboratories should optimize their protocols to accurately detect at least all taxa of central interest by testing and using different techniques.