The composition of the gut microbiota is linked to multiple diseases,including Parkin-son's disease(PD).Abundance of bacteria producing short-chain fatty acids(SCFAs)and fecal SCFA concentrations are reduced in PD.SCFAs exert various beneficial functions in humans.In the interventional,monocentric,open-label clinical trial"Effects of Resistant Starch on Bowel Habits,Short Chain Fatty Acids and Gut Microbiota in Parkinson's Disease"(RESISTA-PD;ID:NCT02784145),we aimed at altering fecal SCFAs by an 8-week prebiotic intervention with resistant starch(RS).We enrolled 87 subjects in three study-arms:32 PD patients received RS(PD+RS),30 control subjects received RS,and 25 PD patients received solely dietary instructions.We performed paired-end 100 bp length metagenomic sequencing of fecal samples using the BGISEQ platform at an average of 9.9 GB.RS was well-tolerated.In the PD+RS group,fecal butyrate concentrations increased significantly,and fecal calprotectin concentrations dropped significantly after 8 weeks of RS intervention.Clinically,we observed a reduction in non-motor symptom load in the PD+RS group.The reference-based analysis of metagenomes highlighted stable alpha-diversity and beta-diversity across the three groups,including bacteria producing SCFAs.Reference-free analysis suggested punctual,yet pronounced differences in the metagenomic signature in the PD+RS group.RESISTA-PD highlights that a prebiotic treatment with RS is safe and well-tolerated in PD.The stable alpha-diversity and beta-diversity alongside altered fecal butyrate and calprotectin concentrations call for long-term studies,also investigating whether RS is able to modify the clinical course of PD.

High-quality DNA extraction is a crucial step in metagenomic studies.Bias by different isolation kits impairs the comparison across datasets.A trending topic is,however,the analysis of multiple metagenomes from the same patients to draw a holistic picture of microbiota associated with diseases.We thus collected bile,stool,saliva,plaque,sputum,and conjunctival swab samples and performed DNA extraction with three commercial kits.For each combination of the specimen type and DNA extraction kit,20-gigabase(Gb)metagenomic data were generated using short-read sequencing.While profiles of the specimen types showed close proximity to each other,we observed notable differences in the alpha diversity and composition of the microbiota depending on the DNA extraction kits.No kit outperformed all selected kits on every specimen.We reached consistently good results using the Qiagen QiAamp DNA Microbiome Kit.Depending on the specimen,our data indicate that over 10 Gb of sequencing data are required to achieve sufficient resolution,but DNA-based identification is superior to identification by mass spectrometry.Finally,long-read nanopore sequencing confirmed the results(correlation coefficient＞0.98).Our results thus suggest using a strategy with only one kit for studies aiming for a direct comparison of multiple microbiotas from the same patients.