Choice of DNA extraction method affects stool microbiome recovery and subsequent phenotypic association analyses.

The lack of standardization in the methods of DNA extraction from fecal samples represents the major source of experimental variation in the microbiome research field. In this study, we aimed to compare the metagenomic profiles and microbiome-phenotype associations obtained by applying two commercially available DNA extraction kits: the AllPrep DNA/RNA Mini Kit (APK) and the QIAamp Fast DNA Stool Mini Kit (FSK). Using metagenomic sequencing data available from 745 paired fecal samples from two independent population cohorts, Lifelines-DEEP (LLD, n = 292) and the 500 Functional Genomics project (500FG, n = 453), we confirmed significant differences in DNA yield and the recovered microbial communities between protocols, with the APK method resulting in a higher DNA concentration and microbial diversity. Further, we observed a massive difference in bacterial relative abundances at species-level between the APK and the FSK protocols, with > 75% of species differentially abundant between protocols in both cohorts. Specifically, comparison with a standard mock community revealed that the APK method provided higher accuracy in the recovery of microbial relative abundances, with the absence of a bead-beating step in the FSK protocol causing an underrepresentation of gram-positive bacteria. This heterogeneity in the recovered microbial composition led to remarkable differences in the association with anthropometric and lifestyle phenotypes. The results of this study further reinforce that the choice of DNA extraction method impacts the metagenomic profile of human gut microbiota and highlight the importance of harmonizing protocols in microbiome studies.

Exome sequencing in patient-parent trios suggests new candidate genes for early-onset primary sclerosing cholangitis.

BACKGROUND & AIMS: Primary sclerosing cholangitis (PSC) is a rare bile duct disease strongly associated with inflammatory bowel disease (IBD). Whole-exome sequencing (WES) has contributed to understanding the molecular basis of very early-onset IBD, but rare protein-altering genetic variants have not been identified for early-onset PSC. We performed WES in patients diagnosed with PSC ≤ 12 years to investigate the contribution of rare genetic variants to early-onset PSC. METHODS: In this multicentre study, WES was performed on 87 DNA samples from 29 patient-parent trios with early-onset PSC. We selected rare (minor allele frequency < 2%) coding and splice-site variants that matched recessive (homozygous and compound heterozygous variants) and dominant (de novo) inheritance in the index patients. Variant pathogenicity was predicted by an in-house developed algorithm (GAVIN), and PSC-relevant variants were selected using gene expression data and gene function. RESULTS: In 22 of 29 trios we identified at least 1 possibly pathogenic variant. We prioritized 36 genes, harbouring a total of 54 variants with predicted pathogenic effects. In 18 genes, we identified 36 compound heterozygous variants, whereas in the other 18 genes we identified 18 de novo variants. Twelve of 36 candidate risk genes are known to play a role in transmembrane transport, adaptive and innate immunity, and epithelial barrier function. CONCLUSIONS: The 36 candidate genes for early-onset PSC need further verification in other patient cohorts and evaluation of gene function before a causal role can be attributed to its variants.