DAG3 is one of Lifelines' additional assessments performed in collaboration with the UMCG department of genetics (see also: DAG1 and DAG2). DAG is the abbreviation of DArmGezondheid, or “Gastrointestinal health” in Dutch.

There is increasing insight into the role of bacterial composition in the intestine and upon the occurrence of (chronic) diseases. Previous research in DAG1 (DEEP) demonstrated the relation of the microbiome and lipids, but also many other diseases and intrinsic and extrinsic factors. The purpose of DAG3 was to gather additional data and biological samples from Lifelines participants to answer research questions such as:

• ‘What is the role of the microbiome in the occurrence of chronic diseases?’
• ‘What is the relation between genetic variants, methylation, gene expression and metabolite levels?’

A total of approximately 9500 participants were included in the DAG3 dataset:

• 700 were children aged 8-17 years
• 400 participants were also included in the (DEEP) dataset
• ~9000 participants were genotyped in the UGLI project.

Metagenomic sequencing of the Lifelines DAG3 samples was performed to assess taxonomy, strain diversity and functionality. Uniquely, not only are all individuals fully genotyped but glycerol aliquots of their microbiota are also stored to enable bacterial culture for further functional studies. Lifelines DAG3 will have the power to study host-microbe interactions and will also allow studies to move from association to causality ¹⁾.

Besides the samples used to generate the microbiome data, questionnaires were also sent to participants to gather phenotypic data on gastrointestinal health symptoms by means of Rome III criteria questionnaire (Longstreth etal 2006) and the Bristol Stool Form Scale (O’Donnell etal 1990).

Participants who were invited for assessment 2A according to the regular Lifelines invitation process were asked to also participate in this study. In addition to the regular 2A measurements, DAG3 participants underwent the following steps:

2A Visit 1

• Signed DAG3 IC available
• Registration of participant
• 1 x throat and 1 x nose swab with amies liquid medium
• 1 x throat and 1 x nose e-nat-swab with preservation solution for DNA-isolation
• Swabs are stored in refrigerator at the visit location and processed/frozen within 48h
• Face-to-face instruction of stool collection
• Hand over written stool collection instructions and collection material

2A Visit 2

• (Additional) puncture blood in PAXgene and registered, stored at room temperature at visit location

Participants received a package with the following materials:

• 3 precoded cups to put stool in by means of a provided pipet
• 2 tubes with amies liquid medium and glycerol to put stool in with provided e-swabs, and two barcoded labels for the tubes
• a nontransparent bag to store the samples in the home freezer immediately after collection
• participants were instructed to fill in a questionnaire and stool diary in the week of stool collection

• Swabs containing liquid amies medium were processed into 3 aliquots, containing glycerol. The processing of swabs to aliquots was performed in a flow chamber to prevent contamination. Aliquots were stored at -80C.
• Stool samples were taken in between 16:30 and 19:00 by hired personnel and transported to the Lifelines lab in refrigerated boxes containing cooling elements. Collectors asked the participants at home if they filled all three cups and two tubes and if not, the reason for an incomplete collection. At the Lifelines lab all cups and tubes were stored at -80C.
• blood samples were collected in a PAXgene tube, by means of a ‘butterfly needle’. A “discard tube” was drawn preceding the PAXgene tube. The PAXgene tube was transported to the Lifelines lab, registered and stored at -80C.

The following additional samples have been collected for DAG3 from approximately 9500 participants:

• 9535 e-nat nose samples
• 28612 nose swab aliquots (glycerol)*
• 9425 throat e-nat samples
• 28245 throat swab aliquots (glycerol)*
• 16827 feces swab samples*
• 25376 feces cryo samples*
• 8931 PAXgene samples

• 5400 complete diaries
• 2482 incomplete diaries (i.e. may miss one or more days)

*Multiple samples from a single participant, sourced from the nose- or throat swab.

• Gacesa, R et al. (2022) Environmental factors shaping the gut microbiome in a Dutch population. Nature 604, 732–739