Qiaamp dna tissue kit

Fresh frozen tissues were collected from each resected nodule. Genomic DNA was extracted using the QIAamp DNA Tissue Kit (Qiagen). The samples were subjected to wide panel‐genomic sequencing (pan‐cancer 1021‐gene panel, Geneplus Technology Inc.) at the coverage depth of 1800×. Germline DNA from peripheral blood mononuclear cells of the same patients was used as a reference. The detection and data analysis were performed as described previously.²², ²³, ²⁴

Non-viable BT was defined as the presence of bactDNA in blood in a negative microbiological culture. Total DNA extraction from homogenized specimens was undertaken with the QIAamp DNA Tissue kit (QIAgen, Barcelona, Spain). A standard PCR followed by partial nucleotide sequencing of the 16SrRNA gene was performed according to the methodology described elsewhere^{51 (link)}. Two microliters of template were added into a reaction mix containing 10 mmol/L Tris buffer (pH 8.3), 50 mmol/L KCl, 1.5 mmol/L Mg₂, 200 μmol/L of each deoxynucleoside triphosphate, 50 pmol of primers 5′-AGAGTTTGAT-CATGGCTCAG-3′ and 5′-ACCGCGACTGCTGCT-GGCAC-3′, and 1.25 U BioTaq (Bioline, London, England) to reach a final volume of 50 μL. The primers located at positions 7–27 and 531–514 (Escherichia coli numbering) are universal eubacterial primers that will amplify any known bacterial 16S ribosomal RNA gene. A 35-cycle PCR was run in a GeneAmp 9700 (Applied Bio-systems, Foster City, CA) at: 94 °C for 30 s, 55 °C for 30 s, and 72°. The detection limit of the technique was 5 pg/mL of bacterial DNA. Samples under detection limit were considered negative.

Genomic DNA was extracted from whole-blood specimens of patients with lung adenocarcinoma using the QIAamp DNA Tissue kit and the QIAamp DNA Blood Mini Kit (Qiagen, Valencia, CA, USA), respectively, according to the manufacturer’s protocols. After preparing the DNA, it was aliquoted and stored at −20 °C and used as templates for the following experiments. Exons 18–21 of EGFR were amplified using a polymer chain reaction (PCR) and then subjected to DNA sequencing as described previously [21 (link)]. The allelic identification of 6 CD44 SNPs (rs1425802, rs11821102, rs10836347, rs13347, rs187115, and rs713330) was examined using the TaqMan SNP genotyping assay and the ABI StepOnePlus Real-Time PCR system (Applied Biosystems, Foster City, CA, USA).

We performed DNA extraction from all field-collected scats using already established approaches described in Modi et al.^{27 (link)}. In brief, we either swabbed twice (samples with no dust) or scraped (samples covered with dust) the top layer of the samples with sterile swabs or blade, respectively. They were lysed overnight in a lysis buffer at 56 °C, and extraction was performed following QIAamp DNA Tissue Kit (Qiagen Inc, Hilden, Germany) protocol. Final elution was performed twice in 100 μl of 1X TE buffer, and the DNA was stored at − 20 °C for long-term use.
We conducted molecular species identification using dhole-specific mitochondrial DholespID-F/R primers described in Modi et al.^{27 (link)}. PCR reactions were performed in 10 µL volumes with 4 µL of hot-start taq mix (Qiagen Inc, Hilden, Germany), 4 µM BSA, 0.5 µM of primer mix and 3 µL of DNA extract. PCR conditions included an initial denaturation (95 °C for 15 min); 50 cycles of denaturation (94 °C for 30 s), annealing (50 °C for 30 s) and extension (72 °C for 35 s); followed by a final extension (72 °C for 10 min). Negative and extraction controls were included to monitor contaminations. Species ascertainment was done through visualization of dhole-specific bands (236 bp) in 2% agarose gel. All the experiments were conducted in Conservation Genetics Lab in Wildlife Institute of India, Dehradun.

Fresh tissues were obtained from each nodule and healthy lung tissue 5 cm from each lesion. Healthy tissues were pathologically confirmed postoperatively. Genomic DNA was extracted from fresh tissues using the QIAamp DNA Tissue Kit (Qiagen, Germany). All the samples were subjected to wide panel-genomic sequencing (pan-cancer 1021-gene panel, Geneplus Technology Inc.) at the coverage depth of 1800 × . DNA from peripheral blood mononuclear cells (PBMCs) of the same patients served as a germline DNA reference. The detection and data analysis were performed as described previously [45 (link), 46 (link)].