DNA isolation of mucosal biopsies and faecal samples

In order to analyse the microbiota composition in biopsy samples or faeces, DNA needs to be extracted from the sample and purified. Mucosal pinch biopsies collected for paper I were snap frozen on liquid N2 and stored on -80°C until DNA isolation analysis.

Biopsies collected in paper II and III were directly snap-frozen on liquid N2 and stored on liquid N2 until DNA isolation analysis. Patients in paper I were also instructed to bring a fresh faecal sample, which were stored directly on -80°C until DNA isolation analysis.

Before DNA isolation of samples for paper I the literature was reviewed before choosing a DNA isolation kit. Previous studies investigating the mucosal microbiota in CD patients using high throughput sequencing techniques had been using a variety of different kits.

For example, the paediatric studies by Gevers et al. 2014 (102) and Haberman et al. 2014 (110) used Qiagen Allprep RNA/DNA kit (Qiagen, Hilden, Germany). Morgan et al. 2012 (101) analysed both faecal and biopsy samples using QIAamp DNA stool mini kit (Qiagen), Chiodini et al. 2015, 2016 and 2018 analysed surgical resection samples from CD patients using DNeasy Powersoil Max (Qiagen) (126, 238, 239). El Mouzan et al.

analysed fungal microbiota in stool and biopsies from paediatric patients using MO BIO Powersoil (MO BIO, San Diego, CA) now sold as DNeasy Powersoil (Qiagen) (129).

Only a few studies had compared relevant DNA isolation kits, and no study had compared the efficiency of the kits on mucosal biopsy samples from the GI tract. Vesty

et al. 2017 compared four DNA extraction methods including MO BIO Powersoil (now sold as DNeasy Powersoil (Qiagen)) and QIAamp DNA Mini Kit (Qiagen) on dental plaque and saliva samples from the oral cavity (240). They evaluated DNA quality and yield as well as bacterial and fungal microbiota structures after 16S ribosomal RNA (rRNA) sequencing of the V3-V4 region. The main findings were that the QIAamp DNA Mini kit produced greater DNA yield compared to the three other methods, but that the kits were similar in terms of bacterial diversity and abundance yield (240).

Since commercial kits used in previous mucosal microbiota papers were all owned by Qiagen, we requested Qiagen support for advice. According to recommendations by the manufacturer, the QIAamp DNA Mini Kit would isolate genomic DNA from patients and gram-negative bacteria, however the lysis procedure was not compatible with isolating gram-positive bacteria. Whereas, DNeasy Powersoil and QIAamp DNA PowerSoil both have a lysis strategy based on bead beating, which would allow isolation of both gram-negative and gram-positive bacteria, including genomic DNA from humans (personal communication). Inclusion of a Proteinase K digestion step after homogenisation with bead beating was also recommended (personal communication).

We chose to use QIAamp PowerFecal (Qiagen) and DNeasy PowerSoil (Qiagen) to isolate DNA from faecal samples and biopsy samples, respectively, in paper I and II.

Manufacturer’s protocol was followed except from the following adjustments; step 3 and 4 in the protocol (vortexing) was replaced by bead-beating using Precellys 24 tissue homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France) at 5000 rpm x 3 rounds of 40 seconds. After bead beating and before centrifuging (Step 5) 20 µL of Proteinase K 20mg/mL were added and samples incubated at 65°C for 30 minutes (paper I and II).

After DNA isolation was performed for paper I, a study comparing DNA isolation kits in faecal samples was published (241), this study compared QIAamp Stool Mini kit and QIAamp PowerFecal. The main findings were that all methods generated adequate DNA

concentrations and DNA quality for sequencing. However, the inclusion of a bead-beating step generated higher microbial diversity and caused increased abundances of gram-positive bacteria (241), further supporting that bead-beating was of importance to regenerate the true microbial composition in the sample. Bead-beating was performed in all studies (I-III).

DNA isolation performed in previous mucosa-associated mycobiota papers varied, but two papers reported to use MO BIO Powersoil (67, 129). A co-author had performed ITS-1 sequencing of faeces from IBD patients (242) using a DNA isolation protocol provided from David Underhill (243) which was designed to destroy the fungal cell wall which is well-known to be challenging to lyse. Therefore, we performed a pilot-study were paired samples from the ileum of IBD patients and HC were isolated with DNeasy PowerSoil (Qiagen) including bead-beating step and proteinase K digestion step and the Underhill protocol including a lyticase treatment step, a bead-beating step and QIAamp DNA Mini Kit (Qiagen). PCR products from 18S rRNA PCR amplification were run on a Bioanalyzer (DNA 1000) chip (Agilent Technologies, Santa Clara, CA). Based on the result's samples isolated with the Underhill protocol harboured more fungal DNA and less contamination and the Underhill DNA isolation protocol was chosen to isolate samples for paper III.

After DNA isolation, the samples for paper I and II were quality tested using a Nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA). Nanodrop uses UV-light to give an estimate of the total DNA concentration (ng/µL) (including all DNA, both human, bacterial, fungal) and the purity of the DNA (260/280 and 260/230 nm ratio).

Since DNA absorb light at around 260 nm, and contaminants at around 280 and 230, a low 260/280 or 260/230 ratio indicate contamination. A DNA concentration above 30 ng/µL together with 260/280 ratio ³1.8 and 260/230 ratio ³2.0 was considered adequate. For paper I-III, DNA in samples were quantified using Qubit (Thermo Fisher Scientific). Qubit is more specific than UV absorbance measurements as the Qubit assay kits only fluorescence when bound to the selected molecule in this case DNA. Qubit can

detect low DNA concentrations. The isolated samples for paper IIII were stored on -80°C until library preparation and amplicon sequencing.