Challenges of next-generation sequencing targeting anaerobes.

Next-generation sequencing allows for investigating the composition of microbiomes that are associated with infection (clinical microbiology) or dysbiosis (microbial ecology). The most commonly applied short-read sequencing technologies are Illumina MiSeq/HiSeq and Ion Torrent PGM, however, other platforms that generate long-reads are under way and optimized. A pre-condition for representative results is an appropriate method for contamination-free collection, homogenization, storage of specimens and a subsequent efficient DNA extraction protocol. As some of the anaerobes such as Clostridia or anaerobe Archaea are robust while others of the same environment, such as spirochetes, possess a very thin cell wall, a chemico-mechanical lysing strategy is recommended but with some precautions to avoid DNA-sheering and overheating. For amplicon sequencing, the Silva-TestPrime online tool helps to find the optimal 16S directed primers for individual studies. For metagenome profiling, the classifier tool has to be selected with helpful decision trees available but a combination based on different strategies seems to be indispensable. Further development of both hard- and software is needed before microbiome results become free of a substantial technology-dependent bias.

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