Location: Horticultural Crops Production and Genetic Improvement Research Unit
Project Number: 2072-21000-057-033-G
Project Type: Grant
Start Date: May 1, 2022
End Date: Oct 31, 2024
Objective:
Primary objective Year 1: Organize volunteer recruitment (to include all necessary administrative approvals) and enroll at least half of the target number (25) of volunteers. Perform the dietary intervention and relevant sample collection.
Secondary objective Year 1: Process samples in small batches and perform gut microbiota (16S amplicon sequencing) profiling on as many samples as possible).
Approach:
Objective 1: Volunteers will be randomly assigned to one of the study arms (placebo or cranberry juice first) and will be provided enough cranberry juice (or placebo when appropriate) for daily consumption over two weeks on their first two visits. Volunteers will be provided with a timetable following the dietary intervention scheme and sampling kits with clear instructions for them to self-sample urine, stool, and a vaginal swab on four time points.
Objective 2: Microbial DNA will be extracted from swabs and from stool samples. DNA quality and quantity will be checked by gel electrophoresis or Nanodrop measurement. For 16S rRNA gene sequencing analyses, universal primers tagged with Illumina adapters and barcodes will be used for PCR from the DNA extracts. PCR will be conducted following standard protocols using a proofreading Taq polymerase. Amplification will be checked by gel electrophoresis, and correct size amplicons will subsequently be subjected to purification. Amplicons concentration will be measured, multiplexed as one sample and sequenced on an Illumina MiSeq. For metagenomic analyses, a subset of samples of interest based on the 16S analyses results will be selected. Genomic DNA concentration and quality will be checked by Qbit and gel electrophoresis. Libraries will be prepared, pooled and sequenced. For all applications, paired-end sequencing will be performed using the Illumina kits. The maximum number of cycles (500 cycles or base) will be required for 16S amplicons, whereas lower read length can be used for metagenomics. HiSeq sequencing will then be undertaken according to Illumina standard protocols. Resulting sequencing files will be analyzed using well validated pipelines. For 16S, initial quality control, removal of adapters, barcodes and primers and de-multiplexing will be performed with Mothur. Operational taxonomic units and amplicon sequence variants will then be determined and classified against reference databases. Basic and multivariate statistical analyses will be performed in PAST. Examples of statistical analyses include for intra-group composition: diversity indices and for intra-group comparisons: PCoA, NMDS (visual representation of the similarity of samples on two axes), ANOVA or non-parametric tests (testing the significance of general/specific differences between groups). Metagenomic data will be subjected to quality control with FastX and assembled with metavelvet. The data will then be uploaded to the MG-RAST online server for comprehensive phylogenetic and functional analysis. For the most part, statistical analyses will be performed directly in MG-RAST, with similar output than for the 16S data. Urine samples will be acidified upon collection and then dilute 50:50 with methanol with 0.1% formic acid, centrifuge, filter through 0.22 µm filters, and inject. Analysis of individual compounds will be performed on a Thermo Scientific TSQ Altis triple quadrapole mass spectrometer with an ESI source. Compound-specific programming in multiple reaction monitoring will be used along with authentic standards of parent phenolics and urinary metabolites and identified, confirmed, and quantified.
