Qiamp dna isolation kit

DNA was isolated from 200 μL of PBMCs from patients and controls using QIAmp DNA isolation Kit (QIAGEN, MD) according to the manufacturer's instructions. The quality of extracted DNA was determined by spectrophotometric analysis. A260/A280 ratio of 1.6 to 2.0 was considered good quality. Integrity of Deoxyribonucleic Acid (DNA) was confirmed by agarose gel electrophoresis. The eluted DNA was stored at −20°C for long term storage.

Patients’ fecal samples were collected in sterile tubes and stored at −80 °C until further processing. The isolation of bacterial DNA was performed using the QIAmp DNA isolation kit following the manufacturer’s instructions (Qiagen, Hilden, Germany). For better cell disruption, we used a mechanical lysis step using the Fast Prep™-24 instrument (MP Biomedicas, Solon, OH, USA) at 6.0 m/s for 2 × 45 seconds. The preparation of amplicon libraries was performed as described previously and sequenced on a MiSeq (2 × 250 bp, Illumina, Hayward, CA, USA) [10 (link)]. The resulting FastaQ files were analyzed using the dada2 package in R (www.r-project.org (version 3.4.2. 2017)). All samples were resampled to equal the smallest library size of 12,448 reads using the phyloseq package and returning 6341 phylotypes. Sequence reads were assigned to a taxonomic affiliation based on the naïve Bayesian classification with a pseudo-bootstrap threshold of 80%. Relative abundances in percentage of phylogenetic ranks, Phylotypes, Family, Order, and Phylum, were used for further analyses. The abundances of those phylotypes with a mean > 1% were compared by the Mann–Whitney test using GraphPad Prism 9. Detected phylotypes were taxonomically assigned to 16 Phyla, 53 Orders, and 77 Families.

According to the manufacturer’s instructions, DNA was isolated from venous blood samples of patients and controls using the QIAmp DNA isolation Kit (QIAGEN, MD). The quality was determined by Nanodrop 2000/Qubit. OD260/280 = 1.8~2.0 was considered good quality. The integrity of Deoxyribonucleic Acid (DNA) was confirmed by agarose gel electrophoresis.
Input DNA is converted to a sequencing library by fragmentation to 100-500bp fragments, the fragments underwent end repair by adding T4 DNA polymerase, then Agencourt AMpure XP magnetic beads were used to obtain the desired short fragment library Beads. Single adenylate A was added to the 3’end and ligation to platform-specific oligonucleotide adapters was done by T4 DNA ligase. The library was then purified and amplified ready for hybridization by Agilent sureselect^XT custom Kit, Cleaning and purification were done using Dynabeads^® MyOne™ Streptavidin T1. The library was again cleaned and quantified by Qubit 3.0 and the Agilent 2100 Bioanalyzer, respectively. The individual library fragments were clonally amplified by PCR; clusters of PCR colonies were then sequenced on the Illumina Hiseq2500 platform to obtain Fast SeQ data. The experimental steps are shown in Figure 1.

72 HBOC family samples used for technical comparisons were identified in the Oncogenetics consultation of the Centre Jean Perrin. 250 HBOC samples used for microfluidic amplification were identified in the Oncogenetics consultation of the Naef K. Basile Cancer Institute (NKBCI) at the American University of Beirut Medical Center (AUBMC, Beirut, Lebanon). DNA was isolated from peripheral blood by standard techniques, using NucleoSpin® Blood XL kit (Macherey-Nagel) at the Centre Jean Perrin and QIAmp DNA isolation kit (Qiagen) at the AUBMC.

Genomic DNA was extracted from a 3 ml EDTA blood sample using the QiAmp DNA isolation kit (Qiagen, GmBH) according to the manufacturer's instructions.