Qiaamp powerfecal pro dna kit

Bacterial DNA extraction was performed using the QIAamp PowerFecal Pro DNA Kit (QIAGEN, Hilden, Germany). For the exhaled breath condensate (EBC), standard protocol was modified as follows: the steps sample preparation, cell lysis, and inhibitor removal technology were omitted. The first step in extracting DNA from EBC samples was, therefore, adding 600 µL of high-concentration salt solution CD3 to an average of 700 µL of the sample. The manufacturer’s instructions were followed from that step (bind DNA, wash, elute). Elution was performed in a volume of 50 µL of C6 solution. DNA extraction of throat swabs was performed according to the manufacturer’s instructions, only no bead tubes were used, and instead of this step, the swab was vortexed in 800 µL of lysis buffer (CD1) and incubated at room temperature for 30 min.

The genomic bacterial DNA was extracted from fecal samples using a QIAamp PowerFecal Pro DNA Kit (Qiagen catalog no: 51804). The procedure for the extraction of DNA from rodent fecal samples has been standardized and published in our earlier studies (Rahman et al., 2023 (link)). Briefly, 200–250 mg of fecal sample was added to the PowerBead Pro Tubes containing 800 μL of CD1 solution and then homogenized using a FastPrep-24™ bead grinder (MP Biomedicals, United States) followed by centrifugation at 15,000 × g for 1 min. Then, 200 μL of CD2 solution was added to the supernatant and centrifuged for 1 min. The supernatant was again transferred to a new tube containing 600 μL of CD3 solution, passed through the MB spin column, and subsequently washed by 500 μL of EA and C5 solution. The column was then placed into a new tube, and 50–100 μL of solution C6 was added to the center of the white filter membrane. The DNA was collected after centrifugation at 15,000 × g for 1 min.

Microbial genomic DNA was extracted from the stool samples using the QIAamp PowerFecal Pro DNA Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s protocol. Fecal samples were collected and frozen immediately at −80 °C until DNA extraction. The isolated bacterial material was quantified and characterized using a spectrophotometer NanoDrop™ 2000 (Thermo Fisher Scientific, Inc., Waltham, MA, USA).

Total DNA extractions from faeces were performed using the QIAamp PowerFecal Pro DNA kit (Qiagen, Antwerp, Belgium) according to the type of sample and the manufacturer’s recommendations.
With total bacterial DNA extracted from 83 samples, 16S rDNA profiling targeting the V1-V3 hypervariable region was performed as described previously [25 (link),26 (link)]. All libraries were run with Illumina MiSeq Technology (Illumina, SY—410-1003). The protocol used was the same as that described in the work of Ngo et al. (2018) [26 (link)]. Sequence reads were processed using the MOTHUR software package V1.41.1 and Vsearch for chimera detection [27 (link),28 (link)]. After cleaning and searching chimeras, 13,314,296 sequence reads were obtained, subsampled at 10,000 reads per sample and clustered into 129,317 OTUs (clustering threshold of 0.03). Reference alignment and taxonomic assignments were based on the SILVA database (v1.38.1) [29 (link)] of the full-length 16S rDNA sequences. The file was processed to classify the data into 536 phylotypes at the genus level. Sequencing libraries are available in the GenBank repository under the PRJNA924547 bioproject.

A QIAamp PowerFecal Pro DNA Kit (Qiagen, Hilden, Germany) was employed to extract genomic DNA from the KD337-16^T strain. Subsequently, the SQK-LSK109 Ligation Sequencing Kit on a PromethION Flow Cell (R9.4.1) and Illumina NovaSeq 6000 in paired-end (2 × 151 bp) mode was used for Oxford Nanopore Technologies (ONT) sequencing. After the sequences had been decoded and refined, Flye version 2.8.3 was employed for assembly of the valid ONT sequences. The primary contigs were polished with Racon v1.4.22 and the Illumina read alignment results constructed using Minimap2 v2.17. The DDBJ Fast Annotation and Submission Tool was used to annotate the genome [35 (link)]. Methods described elsewhere [36 (link),37 (link),38 (link)] were employed to quantify the digital DNA–DNA hybridization (dDDH), the amino acid identity (AAI), and average nucleotide identity (ANI). The up-to-date bacterial core genes pipeline (http://leb.snu.ac.kr/ubcg2, accessed on 28 October 2022) [39 (link)] and EDGAR platform were utilized to construct phylogenomic trees [40 (link)], whereas the 3ggNOG 4.5 database and carbohydrate-active enzyme (CAZy) database were employed for functional assignment [41 (link),42 (link)]. The OrthoVenn2 webserver was used for pangenome analysis [43 (link)]. Finally, AntiSMASH software (v. 6.0) was employed to predict putative biosynthetic gene clusters [44 (link)].

DNA was extracted from cecum samples according to the protocol of the QIAamp PowerFecal Pro DNA Kit (Qiagen, Hilden, Germany). The 16s rRNA gene library was constructed according to Illumina’s instructions [40 ]. The final PCR product was sequenced by Macrogen (Seoul, Korea) using a Miseq sequencer (Illumina Inc., San Diego, CA, USA). In Quantitative Insight into Microbial Ecology (QIIME 2), sequence reads were preprocessed, demultiplexing, trimming, and truncating low-quality regions. In addition, taxonomy assignment based on Greengenes reference and rooted/unrooted phylogenetic trees was conducted. Further data analysis and visualization were performed in the R phyloseq package [41 (link)] and GraphPad Prism (version 8.3.0; GraphPad software Inc., San Diego, CA, USA). Phylogenetic distances between groups were calculated from the generalized UniFrac (GUniFrac) [42 (link)]. The negative binomial distribution in differential abundance in comparison with the DSS group was determined using the DESeq2 package (BaseMean > 1 and adjusted p < 0.05) [43 (link)]. To find colitis-associated bacterial taxa, the genera belonging to the differential abundance in DESeq2 analysis were agglomerated at a genus level. Subsequently, Pearson correlation analysis of the bacterial taxa and inflammation biomarkers was carried out.