Qiaamp ucp pathogen dna kit

The nucleic acid extraction, library preparation and sequencing were carried out as previous study.^{17 (link)} Briefly, approximately 3–5 mL peripheral blood was collected, and plasma was obtained by a centrifugation at 4 °C, 1600 rpm, for 10 min. Plasma DNA and RNA were extracted by using QIAamp^® UCP Pathogen DNA Kit and QIAamp^® Viral RNA Kit (Qiagen) according to the manufacturer’s instructions, respectively. To include negative controls, PBMC samples (10⁵ cells/mL) obtained from healthy donors were prepared and underwent the same protocol and processing conditions as the test sample materials. Additionally, sterile deionized water was included as a non-template control (NTC) alongside the specimens. Finally, to help to differentiate pathogenic microorganisms from background microorganisms, a comprehensive list of suspected background or contaminated microorganisms were included at the end of the reports. Nextera XT DNA Library Prep Kit (Illumina) was used to construct libraries for sequencing (Illumina Nextseq CN500).

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, Hilden, Germany Sigma, St. Louis, Missouri) and Tween20 (Sigma) (Amar et al., 2021 (link)). Libraries were constructed using a Nextera XT DNA Library Prep Kit (Illumina, San Diego, CA, United States) (Miller et al., 2019 (link)). The library was quality-assessed by the 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 CN500 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 (NTCs) (Miller et al., 2019 (link)).

All samples were extracted with QIAamp^® UCP Pathogen DNA Kit (Qiagen). Tween 20 and Benzonase (Qiagen) are used to remove DNA from human samples according to the manufacturer’s instructions (Amar et al., 2021 (link)). A QIAamp^® Viral RNA Kit was used to extract RNA, and a Ribo-Zero rRNA Removal Kit (Illumina) was used to remove ribosomal RNA. cDNA was synthesized with reverse transcriptase and dNTP (Thermo Fisher). The construction of the selected DNA and cDNA library was done using Nextera XT DNA Library Prep Kit (Illumina, San Diego, CA) (Miller et al., 2019 (link)). Quality control for the library was performed using a Qubit dsDNA HS Assay Kit and a High Sensitivity DNA kit (Agilent) on an Agilent Bioanalyzer 2100. Then, the library pools were loaded onto an Illumina NextSeq CN500 sequencer for 75 cycles of single-end sequencing of 20 million reads for each library. Negative control (NC) is a peripheral blood mononuclear cell sample of 105 cells/mL prepared alongside each batch with the same protocol (Amar et al., 2021 (link)), and sterile deionized water was used as a nontemplate control (NTC) alongside the specimens (Li et al., 2018 (link)).

DNA was extracted from all BALF samples using a QIAamp^® UCP Pathogen DNA Kit (Qiagen). Human DNA was removed using Benzonase (Qiagen) and Tween20 (Sigma). Total RNA was extracted with a QIAamp^® Viral RNA Kit (Qiagen), and ribosomal RNA was removed by a Ribo-Zero rRNA Removal Kit (Illumina). cDNA was generated using reverse transcriptase and dNTPs (Thermo Fisher). Libraries were constructed for the DNA and cDNA samples using a Nextera XT DNA Library Prep Kit (Illumina, San Diego, CA). Library was quality-assessed by Qubit dsDNA HS Assay kit followed by a high-sensitivity DNA kit (Agilent) on an Agilent 2100 Bioanalyzer. Library pools were then loaded onto an Illumina Nextseq CN500 sequencer for 75 cycles of single-end sequencing to generate approximately 20 million reads for each library.

Bronchoscopy was performed according to standard procedures using a flexible fiberoptic bronchoscope. A special collector was used to collect 3–5 ml of BALF, which was stored at 4°C. The BALF was sent for mNGS analysis (DNA and RNA). DNA was extracted using a QIAamp^® UCP Pathogen DNA Kit (Qiagen), following the manufacturer’s instructions. Human DNA was removed using benzonase (Qiagen) and Tween20 (Sigma). Total RNA was extracted using a QIAamp^® Viral RNA Kit (Qiagen). Ribosomal RNA was removed using a Ribo-Zero rRNA Removal Kit (Illumina). Complementary DNA (cDNA) was generated using reverse transcriptase and deoxynucleoside triphosphates (Thermo Fisher Scientific). Libraries were constructed for DNA and cDNA samples using the Nextera XT DNA Library Prep Kit (Illumina). Library quality was assessed using the Qubit dsDNA HS Assay Kit, followed by a high-sensitivity DNA Kit (Agilent) on an Agilent 2100 bioanalyzer. Library pools were then loaded onto an Illumina NextSeq CN500 sequencer for 75 cycles of single-end sequencing, generating approximately 20 million reads per library. For negative controls, we prepared peripheral blood mononuclear cell samples (10⁵ cells/ml) from healthy donors, in parallel with each batch, using the same protocol. Sterile deionized water was extracted alongside the specimens to serve as a non-template control.

BALF were collected from patients according to standard procedures. DNA was extracted using a QIAamp^® UCP Pathogen DNA Kit (Qiagen) following the manufacturer's instructions and 600 μL of the processed specimens was mixed with glass beads of 0.1–0.2 mm diameter. A vortex mixer (Crystal, TX, United States) was used to disrupt the bacterial cell wall at 1,600 g for 10 min. The tubes were then heated at 99°C for 10 min before DNA extraction. Human DNA was removed using Benzonase (Qiagen) and Tween20 (Sigma) (16 (link)). The differential lysis method was used to remove host DNA. we first use physical hypotonic lysis and chemical lysis to break human cells, and then obtain microbial cells by enzymatic hydrolysis, followed by wall breaking and nucleic acid extraction. Total RNA was extracted with a QIAamp^® Viral RNA Kit (Qiagen) and ribosomal RNA was removed by a Ribo-Zero rRNA Removal Kit (Illumina). The concentration of extracted DNA/RNA was measured using a Qubit Fluorometer before library preparation. cDNA was generated using reverse transcriptase and dNTPs (Thermo Fisher).