Shotgun metagenomic sequencing

Shotgun metagenomic sequencing, or the untargeted sequencing of all microbial genomes present in a specimen, is considerably richer in providing information than amplicon-based profiling approaches. Unlike amplicon-based sequencing, where specific primers are targeted to regions of ribosomal RNA genes, DNA is prepared for shotgun metagenomics by random fragmentation, addition of barcoded sequencing tags, and limited cycle amplification (Figure 1). Since shotgun metagenomics captures a greater variety of gene content in a sample, multi-kingdom compositions at strain-level resolution (as depicted in Figure 2, adapted from (Oh et al., 2014), as well as functional profiles for communities are captured. Shotgun metagenomics have provided key insights into the skin microbiome in atopic dermatitis, including the role of strain-level variation of Staphylococcus aureus (Byrd et al., 2017), and mechanistic understanding of how microbial metabolic pathways are altered to enhance ammonia production and increase skin pH (Chng et al., 2016).

Two different analytical approaches are used for shotgun metagenomic datasets: assembly-based and read-based profiling (for a comprehensive discussion, the authors recommend (Quince et al., 2017). While read-based, assembly-free profiling is faster and mitigates issues with assembly, it relies upon reference genomes at the expense of uncharacterized microbes that have no references available. A popular tool to generate taxonomic profiles without assembly is MetaPhlan, which maps shotgun reads to reference marker genes (Segata et al., 2012). These data may then be used to derive the alpha and beta diversity metrics previously described. Functional profiles can be produced using the HUMAnN tool (Abubucker et al., 2012) or similar, that takes the DNA reads and maps them against universal gene-protein databases. This allows identification of the proteins encoded by the DNA and functional pathway linkage of the proteins.