An optimized approach to the metagenomic population studies: from sample donation to community profiling

In this study, we intended to establish an optimized approach to investigating the metagenomes of fecal samples. We checked how experimental procedures on different stages of the analyses influenced community profiling and investigated the bias caused by subsequent bioinformatics procedures.We designed a test experiment that allowed us to scrutinize each part of the investigation procedure of fecal samples metagenomes. We started with the DNA isolation procedure and finished with computational methods for microbial communities profiling. For this study, we used: three fecal samples obtained from healthy volunteers (S1, S2, S3) and two commercially available bacterial mixes (ATCC Bacterial Mix MSA-2006 - a mix of 12 known bacteria and ATCC Genomic DNA MSA-1003 - a mix of 20 bacterial DNA). We tested three different times of homogenization: 10 min., 15 min., 20 min. We prepared DNA libraries using three sets of reagents from leading manufacturers - KAPA, Nextera, and Qiagen, obtaining 36 libraries. Additionally, we also introduced ATCC Genomic Mix DNA isolate sample to imitate the perfect isolation serve, giving three additional libraries (for KAPA, Nextera, and Qiagen). In total, we prepared 39 libraries - 13 libraries with each kit.