Normalization and calibration in the metagenomic analysis of CF lung microbiomes

Abstract

Next-generation sequencing is becoming increasingly affordable, which makes whole-metagenome sequencing an attractive alternative to traditional 16S rDNA, RFLP, or culturing approaches to analysis of metagenomic samples. The advantage of whole-metagenome sequencing is a broader spectrum of detectable microorganisms (including also fungi and many viruses), quantitative analysis of the microbial community and information about the metabolic capacity and physiological features of the studied metagenome, even without the knowledge of genotypes and phenotypes of all members of the microbial community. Problems of 16S rDNA sequencing, such as unknown copy number of the 16S gene and lack of sequence homology to some of the target 16S genes are not present in whole-metagenome sequencing. On the other hand, next-generation sequencing suffers from biases resulting in non-uniform coverage of the sequenced genomes (e.g. GC- bias, sequence based differences in ligase efficiency), differences in the genome length of microorgansms and their content of variable genomic elements (e.g. gene islands, phages), which, however, can and must be normalized.