ena-STUDY-ROSWELL PARK CANCER INSTITUTE-25-11-2018-05:25:12:138-948
Oral, respiratory airway, and lung tissue bacterial microbiomes in stage I non-small cell lung cancer
In this study, 16S sequencing was performed to assess the bacterial microbiomes in DNA isolated from pre-operative saliva and bronchial lavage fluid samples, and surgically resected tumor and adjacent normal lung tissues of patients with pathologic stage I non-small cell lung cancer. Samples were obtained from the Lung Cancer Biospecimen Resource Network, University of Virginia, VA, USA. For the samples procured by the repository, normal saline was used for collection of 20-40 ml of bronchial lavage fluids, which were centrifuged at 1,300 g for 25 minutes, following which pellets were resuspended in 1 ml of the supernatant and stored at -80 ºC or in liquid nitrogen. Salivette tubes (Sarstedt, Newton, NC) were used for collecting saliva which were similarly stored. DNA and RNA were separately extracted from the same tissue samples using TissueLyser II, 5-mm stainless steel beads, and AllPrep DNA/RNA/Protein Mini kit (Qiagen, Valencia, CA). QIAamp UCP Pathogen Mini kit and Pathogen Lysis L tubes (Qiagen) were used to extract DNA from bronchial lavage fluid (0.4 ml) and saliva (0.2 ml). The protocol "Pretreatment of Pathogen DNA from 400 µl Whole Blood (Mechanical Pre-lysis Protocol)" provided with the kit was used. The kit's ATL buffer with reagent Dx was added to samples to make their volume to 0.4 ml before mechanical lysis. The two types of samples were processed in separate batches, and each batch included a negative control for DNA extraction, an empty, sterile, nuclease-free 1.5-ml microcentrifuge tube. DNA was also isolated in each of the two batches from a positive control, ZymoBIOMICS Microbial Community Standard (product D6300, Zymo, Irvine, CA). 16S sequencing of isolated DNA, involving 16S PCR, sequencing library generation, and library sequencing, was performed in four batches. All samples of a patient were sequenced in the same batch, and each batch included a no-template negative control for 16S PCR. The 16S sequencing method suggested in the 16S Metagenomic Library preparation guide of Illumina (San Diego, CA) was followed. An ~464 bp amplicon of bacterial 16S rRNA V3-V4 region was amplified with forward and reverse primers of sequences TCGTCGGCAGCGTC-ad-CCTACGGGNGGCWGCAG and GTCTCGTGGGCTCGG-ad-GACTACHVGGGTATCTAATCC, respectively (ad = AGATGTGTATAAGAGACAG). DNA (25 ng) was subjected to 35 cycles of PCR with annealing temperature of 55 ºC and KAPA HiFi HotStart DNA polymerase. For samples for which 25 ng DNA was unavailable, the maximum volume of a sample's DNA preparation was used. Amplified DNA was purified with AMPure XP beads and was indexed with Nextera XT index kit in an 8-cycle PCR to generate sequencing libraries. Libraries were purified with AMPure XP beads and 12 pmoles of each library was sequenced on a MiSeq instrument (Illumina) with v3 sequencing reagents to obtained paired 300 bp reads. Between 46 and 48 libraries were multiplexed in each sequencing run. A PhiX library was spiked in at 10% molarity. Separately, tumor tissue RNA isolates were subjected to RNA sequencing (ENA study PRJEB29932).
In this study, 16S sequencing was performed to assess the bacterial microbiomes in DNA isolated from pre-operative saliva and bronchial lavage fluid samples, and surgically resected tumor and adjacent normal lung tissues of patients with pathologic stage I non-small cell lung cancer. Samples were obtained from the Lung Cancer Biospecimen Resource Network, University of Virginia, VA, USA. For the samples procured by the repository, normal saline was used for collection of 20-40 ml of bronchial lavage fluids, which were centrifuged at 1,300 g for 25 minutes, following which pellets were resuspended in 1 ml of the supernatant and stored at -80 ºC or in liquid nitrogen. Salivette tubes (Sarstedt, Newton, NC) were used for collecting saliva which were similarly stored. DNA and RNA were separately extracted from the same tissue samples using TissueLyser II, 5-mm stainless steel beads, and AllPrep DNA/RNA/Protein Mini kit (Qiagen, Valencia, CA). QIAamp UCP Pathogen Mini kit and Pathogen Lysis L tubes (Qiagen) were used to extract DNA from bronchial lavage fluid (0.4 ml) and saliva (0.2 ml). The protocol "Pretreatment of Pathogen DNA from 400 µl Whole Blood (Mechanical Pre-lysis Protocol)" provided with the kit was used. The kit's ATL buffer with reagent Dx was added to samples to make their volume to 0.4 ml before mechanical lysis. The two types of samples were processed in separate batches, and each batch included a negative control for DNA extraction, an empty, sterile, nuclease-free 1.5-ml microcentrifuge tube. DNA was also isolated in each of the two batches from a positive control, ZymoBIOMICS Microbial Community Standard (product D6300, Zymo, Irvine, CA). 16S sequencing of isolated DNA, involving 16S PCR, sequencing library generation, and library sequencing, was performed in four batches. All samples of a patient were sequenced in the same batch, and each batch included a no-template negative control for 16S PCR. The 16S sequencing method suggested in the 16S Metagenomic Library preparation guide of Illumina (San Diego, CA) was followed. An ~464 bp amplicon of bacterial 16S rRNA V3-V4 region was amplified with forward and reverse primers of sequences TCGTCGGCAGCGTC-ad-CCTACGGGNGGCWGCAG and GTCTCGTGGGCTCGG-ad-GACTACHVGGGTATCTAATCC, respectively (ad = AGATGTGTATAAGAGACAG). DNA (25 ng) was subjected to 35 cycles of PCR with annealing temperature of 55 ºC and KAPA HiFi HotStart DNA polymerase. For samples for which 25 ng DNA was unavailable, the maximum volume of a sample's DNA preparation was used. Amplified DNA was purified with AMPure XP beads and was indexed with Nextera XT index kit in an 8-cycle PCR to generate sequencing libraries. Libraries were purified with AMPure XP beads and 12 pmoles of each library was sequenced on a MiSeq instrument (Illumina) with v3 sequencing reagents to obtained paired 300 bp reads. Between 46 and 48 libraries were multiplexed in each sequencing run. A PhiX library was spiked in at 10% molarity. Separately, tumor tissue RNA isolates were subjected to RNA sequencing (ENA study PRJEB29932).