Qubit dsdna hs assay kit

DNA was extracted from all samples using a QIAamp® UCP Pathogen DNA Kit (Qiagen) following the manufacturer’s instructions. Human DNA was removed using Benzonase (Qiagen) and Tween20 (Sigma) [6 (link)]. 10 nanograms DNA samples were used for library construction through Nextera XT DNA Library Prep Kit (Illumina, San Diego, CA) [7 (link)]. Library was qualitatively assessed by Qubit dsDNA HS Assay kit, followed by High Sensitivity DNA kit (Agilent) on an Agilent 2100 Bioanalyzer. Library pools were then loaded onto an Illumina Nextseq 550Dx sequencer for 75 cycles of single-end sequencing to generate approximately 20 million reads for each library. For negative controls, we also prepared peripheral blood mononuclear cell(PBMC) samples with 10⁵ cells/mL from healthy donors in parallel with each batch, using the same protocol, and sterile deionized water was extracted alongside the specimens to serve as non-template controls (NTC) [7 (link), 8 (link)]. DNA-free water went through DNA extraction and mNGS analysis as a blank control group to assess the degree of background contamination associated with DNA extraction kit and sequencing reagents together.

RNA-seq libraries were constructed from 500 ng RNA of each sample using KAPA RNA Hyper kit with RiboErase following the manufacturer’s instructions. Different adaptors were used for multiplexing samples in one sequencing run. Library concentrations and quality were examined by Qubit ds DNA HS Assay kit and Agilent 4150 Tapestation system (Agilent, Santa Clara, CA). The libraries were pooled and diluted to 2 nM in elution buffer (10 mM Tris-HCL, pH 8.5) and then denatured using the Illumina protocol. The denatured libraries were diluted to 10 pM by pre-chilled hybridization buffer and loaded onto Illumina NextSeq 500 (Illumina, San Diego, CA) and run for 75 cycles using a single-read recipe according to the manufacturer’s instructions. The data quality examination and demultiplexing procedure were implemented with Illumina SAV and Illumina Bcl2fastq2 version 2.17 program, respectively. TopHat (version 2.0.13)^{64 (link)} was applied to align sequencing reads to the human reference genome assembly GRCh37 (hg19) for unique alignments. RefSeq transcript annotations were obtained from the UCSC Genome Browser^{65 (link)}, and read fragments aligned to known exons were counted per gene using Htseq (version 0.6.1p1)^{66 (link)}. All analyses were conducted at the gene level. The RNA-seq raw counts were normalized to the transcripts per million (TPM) unit for the following analyses.

CAGE libraries were prepared as described by Takahashi et al. (25 (link)) using 5 μg of total RNA as input material per sample. Samples were individually barcoded using the sequences outlined by Takahashi et al. (25 (link)) with each pooled library containing eight unique barcoded samples (the following barcodes were used: CTT, GAT, ACG, ATG, TAG, TGG, GTA, and GCC). The pooled libraries were quantified using the Qubit 2.0 Fluorometer (Thermo Fisher Scientific) with the Qubit dsDNA HS Assay Kit and assessed for quality and fragment size on the Agilent 2100 Electrophoresis Bioanalyser Instrument (Agilent Technologies) with the DNA HS Kit before being sent for sequencing on a high-throughput run on the Illumina HiSeq 2500 System (Illumina) at the Centre for Genomics Research, University of Liverpool.

Total RNA was extracted using TRIzol™ Plus RNA Purification Kit following owing manufacturer’s recommendations. RNA concentration was measured using Qubit® RNA HS Assay Kit in Qubit® 2.0 Flurometer (Invitrogen) and RNA integrity was assessed using the RNA Agilent RNA 6000 Pico Kit of the Bioanalyzer 2100 system (Agilent Technologies). For the synthesis of complementary DNA (cDNA), the SuperScript VILO MasterMix (Invitrogen) was used for the first strand, while the NEBNext mRNA Second Strand Synthesis Module (New England Biolabs) was used for the second strand. Then, cDNA was purified using AMPure beads. Sequencing libraries were generated using Nextera XT DNA Library Preparation Kit for Illumina®, following manufacturer’s recommendations. The library quantification was performed with the Qubit® dsDNA HS Assay kit, and the fragment sizes were analyzed using the High Sensitivity DNA Analysis Kit on the Bioanalyzer 2100 (Agilent Technologies). Following this, sequencing was performed on an Illumina® NextSeq 500 platform using the NextSeq 500/550 High Output v2.5 (300 cycles) kit and paired-end reads were generated.

The extraction of DNA was carried out from rhizosphere soil samples and the bulk soil using the DNeasy PowerSoil Pro kit (Qiagen, Hilden, Germany) following the instruction protocol. Samples were allowed to thaw; 0.25 g was weighed from each soil sample for DNA extraction. All the datasets used in this study are from the shotgun method of whole–metagenome sequencing. This was done at MR DNA, Shallowater, TX, USA. A life technologies assay kit-Qubit^® dsDNA HS was used to obtain samples’ DNA concentration. The libraries were prepared according to the manufacturer’s instructions using the Illumina DNA Prep (M) tagmentation library preparation kit. The libraries were made with 20–50 ng of DNA. The samples were fragmented and adapter sequences were added. These adapters were used in a limited-cycle PCR in which the material was supplemented with unique indices. The ultimate concentration of the libraries followed the library preparation. After library preparation, the libraries’ final concentrations were assessed using the Life Technologies Qubit^® dsDNA HS Assay Kit, and the average library size was estimated using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). After that, the libraries were pooled at 0.6 nM equimolar ratios and sequenced at paired end for 300 cycles on the NovaSeq 6000 system (Illumina, San Diego, CA, USA).

An Oncomine Colon cfDNA Assay (Thermo Fisher Scientific) was used to generate libraries from the cfDNA, following the manufacturer’s instructions. The purity and concentration of the library were assessed using the Qubit dsDNA HS Assay kit and the Agilent 2100 Bioanalyzer, respectively. Unique index tags were added to each DNA fragment during library preparation. The Ion Chef System and the Ion 530 Kit-Chef were used for template preparation, followed by sequencing on the Ion S5 system using Ion 530 chips. A six-plex library pool was applied to the Ion 530 chip. We used a cfDNA panel covering 14 genes and 48 amplicons, including 240 hot spots (SNVs and short indels). The genes included in the panel were KRAS, TP53, APC, FBXW7, GNAS, MAP2K1, CTNNB1, ERBB2, PIK3CA, BRAF, EGFR, SMAD4, NRAS, and AKT1. Clean reads were mapped to the human reference genome (hg19). A variant caller was used to filter and call mutations in targeted regions of each gene [20 (link), 21 (link)]. The limit of detection for each variant (mutant allele frequency [MAF]) was 0.15%. The average coverage ranged from 20,000x to 50,000x.

Chromatin was quantified using Qubit dsDNA HS (High Sensitivity) assay kit and Qubit™ 3 Fluorometer (Invitrogen). Two biological replicates were prepared for sequencing. For library preparation, NEBNext^® ChIP-Seq Library Prep Master Mix Set for Illumina^® (E6240, NEB) and NEBNext^® Multiplex Oligos for Illumina^® were used according to the manufacturer’s protocol. Libraries were quantified using Qubit dsDNA HS assay kit and quality and fragment distribution were examined with Agilent High Sensitivity DNA kit. Sequencing was performed on the NextSeq500 (HighOutput SR75) in Lexogen, BioCenter in Vienna, Austria.