Next-generation sequencing library preparation for 16S rRNA microbiome analysis after serpin treatment

Abstract

Serine protease inhibitors, serpins, can have profound effects on many systems in the body including immune defense systems. The microbiome, specifically the gut and lung bacterial microbiota, is now known, under some conditions, to alter immune defenses. DNA library preparation for microbiome studies is a procedure that prepares microbial genomic DNA to be sequenced in next-generation sequencing platforms. The construction involves a PCR reaction that will amplify the 16S rRNA gene and will incorporate a specific index and adaptors to the fragments. After confirmation of the product amplification by gel electrophoresis, samples will be later normalized to the same DNA amount of 240 ng. Final concentration of the library is validated by quantitative PCR (qPCR). Here we describe methods to analyze changes in the microbiome after treatment with immune-modulating agents, specifically serpins.