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 9500 participants were included in the DAG3 dataset:

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

Metagenomic sequencing of the Lifelines DAG3 samples is underway 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 GI health symptoms by means of Rome III criteria questionnaire (Longstreth etal 2006) and the Bristol Stool Form Scale (O’Donnell etal 1990).

This protocol is approved by the METc of the University Medical Centre in Groningen (UMCG) in October 2007, number METc 2007/152. This amendment concerns the collection of additional biomaterial and data among a subset of 10.000 adult Lifelines participants in the age of 8 and older. Numbers and age of respondents cannot be guaranteed by Lifelines. Participants are invited for the second Lifelines screening according to the regular Lifelines invitation process, and asked to also participate in this study. This means that participants have to collect feces at home. Furthermore, 4 swabs will be collected at a Lifelines location during the first visit to the Lifelines location, and additional blood (1 tube PAXgene) will be collected at a Lifelines location during the second visit. Swabs concern two of the throat and two of the nose. Participants are allowed to refuse part of the sample collections, i.e. feces, swabs or Paxgene. Further details are provided on the following pages.

From the start of the study, Lifelines participants who are invited to come to a Lifelines location for the second Lifelines screening, will also receive information about the present add-on study and a link to detailed information on DAG3 on the Lifelines website. Participants who want to participate in DAG3 can tick the box for participation DAG3 on the availability form of the 2e Lifelines screening. When participants agree to participate in DAG3 they receive a letter and an Informed Consent form to sign and bring to Lifelines during their first visit. IC’s will be brought to the Bloemsingel with the regular daily transport. At the scanroom IC’s will be scanned and registered. From the start of the study only participants who participate in a digital way will be invited, including 6 Lifelines locations (i.e. Sneek, Leeuwarden, Drachten, Groningen, Oude Pekela, Emmen). If the participating group appears to be skewed according to age, gender or socio-economic status , it can be decided to also include participants who will be invited by paper, and fill out paper versions of the diary. Besides response rates, additional information on number of samples collected per participant etc. will be monitored, to be able to adjust activities when needed. Invitations will be sent until 10.000 participants have agreed to participate. In the information folder, which will be placed on the Lifelines website, it is clarified that participants who agree to participate will receive an instruction for stool collection and stool collection material during the first visit at the Lifelines location. Furthermore, they are informed about the procedure regarding the additional blood collection during the second Lifelines visit of the second screening.

First visit: * Signed IC available * Register participation in Utopia * Four swabs taken and registered and stored in refrigerator * Instruction stool collection * Hand over instruction and stool collection material

Second visit: * (Additional) puncture blood in PAXgene and registered, stored by room temperature at location

Participants who agreed to participate are known to physician assistants in the Lifelines agenda. Physician assistants use this agenda to register the visits and measurements of participants to Lifelines locations. During the first visit, participants will get an instruction on the stool collection to be collected at home. At home, participants put their stool sample in 3 precoded cups by means of a pipet and in 2 tubes with an e-swabs. Two barcoded labels are included in the stoolmaterials to attach to the unlabeled e-swabs tubes. E-swab tubes include amysmedium and glycerol. All stool samples are stored in a nontransparent bag in the freezer directly. Furthermore, two swabs will be taken during the first visit, 1 x throat, and 1 x nose, which include amysmedium. In addition additional e-nat-swabs of throat and nose, which includes another presercation solution for DNA-isolation will te taken. All 4 swabs will be stored in the refrigerator at the locations, and transported the next day with the regular Lifelines transport to the Lifelines lab in Groningen. On Friday, no DAG3-participants will be seen for the first visit, since swabs have to be processed within 48 hours. At the Lifelines lab the 4 swabs for dna isolation will be registered and put into the freezer. Swabs containing amysmedium will be processed into 3 aliquots, containing glycerol. The preparation of glycerol will be performed by the Microbiology department and transported in 250 ml bottles to Lifelines. The processing of swabs to aliquots will be performed in a flowkast to prevent contamination. Aliquots will be stored at -80.

When stool samples are taken in, the coded bag is registered and transported in cool boxes containing cooling elements. Hired personnel from Randstad will take stool samples at participants’ homes, and transport it in the evening to the Lifelines lab. They will ask participants at home if they filled both three cups, and two swabs. And if not, the reason for an incomplete collection. At the Lifelines lab both the cups and the swabs have to be registered in BIMS and stored in the -80 freezers at the Lifelines lab at the Bloemsingel. The next day, the final registration will take place and feces samples will be stored at CFB.

Ten workdays after the visit to Lifelines, participants receive an e-mail and are invited to fill in a stooldiary and a short questionnaire, to be accessed by a link in the mail. Participants are asked to fill out the diary every day during the week, in which they collect their stool samples. A few (minimal 2) days later, participants are called by Lifelines personnel from the Service Bureau in Bedum, to make an appointment for the transport of stoolsamples from participants homes to the Lifelines lab. In an excel file, for every participating location specific days will be selected for picking up the stool samples, to be able to collect as many stool samples at one day as possible, concentrated in a small region, to work efficiently, within small distances. To work efficiently stool samples will be taken in from 16.30 until 19.00, times at which most respondents are at home. If possible, participants receive an sms to remind them on the stool collection and date of picking up the stool sample. Furthermore, they are reminded that stool samples should be put in the freezer directly, including the swabs they have to take of their feces. In addition, it is explained that the diary has to be filled out in the same week participants collect their stoolsample. If participants have any questions about the stool collection, the appointment, or the diary they can contact the phone number they receive in the information folder, which is of the Service Bureau in Bedum. To be able to answer questions on the add-on study, employees of the Service Bureau will be trained by Lifelines/Genetics personnel.

After the regular venapuncture of the Lifelines screening, participants who agreed to participate in the add-on study will have an additional puncture for a blood collection in a PAXgene tube, by means of a ‘butterfly needle’. An empty dummy tube will precede the Paxgene tube. The dummy tube can be thrown away, and the Paxgene tube will be transported (KT) to Lifelines lab, registered and stored in -80 freezers.

It is not desirable to perform two punctures among children. Since children already are punctured by means of butterfly needle, this seems to be no problem, Paxgene can be collected after the regular tubes.

DAG3 e-nat nose samples 9535 DAG3 nose swab samples 9540 DAG3 nose swab aliquots (glycerol) 28612 DAG3 throat e-nat samples 9425 DAG3 throat swab samples 9421 DAG3 throat swab aliquots (glycerol) 28245 DAG3 feces swab samples 16827 DAG3 feces cryo samples 25376 DAG3 paxgene samples 8931