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"bioproject": "PRJEB34172",
"samples-count": 71,
"accession": "MGYS00006271",
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"last-update": "2023-08-02T17:40:41",
"secondary-accession": "ERP117039",
"centre-name": "ROSWELL PARK CANCER INSTITUTE",
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"study-abstract": "In this study, 16S sequencing was performed to assess the bacterial microbiomes in DNA isolated from pre-operative bronchial lavage fluid samples of patients with pathologic stage I non-small cell lung cancer. Thirty-five of this study's bronchial lavage fluid samples were independently processed for DNA isolation and 16S sequencing using the same methods as this study's in a past study whose data are available in the EBI ENA repository with accession number PRJEB29934 (patient identifiers used in both studies are the same). 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 supernatants were collected and stored at -80 oC or in liquid nitrogen.QIAamp UCP Pathogen Mini kit and Pathogen Lysis L tubes (Qiagen) were used to extract DNA from bronchial lavage fluid (0.4 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 study included 3 negative controls for DNA extraction, an empty, sterile, nuclease-free 1.5-ml microcentrifuge tube. DNA was also isolated in duplicate from a positive control, ZymoBIOMICS Microbial Community Standard (product D6300, Zymo, Irvine, CA). Bronchial lavage fluid samples of two patients were mixed 1:1 volume for DNA extraction in duplicate from final 0.4 ml. This was done for two sets of two-patients samples. 16S sequencing of isolated DNA, involving 16S PCR, sequencing library generation, and library sequencing, was performed in one batch. 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. A PhiX library was spiked in at 10% molarity. Multiplexed libraries were sequenced twice using two MiSeq sequencing runs. Sequencing data uploaded to ENA has merged raw (CASAVA de-multiplexed) fastq.gz files of the two runs.",
"study-name": "Respiratory airway microbiomes in stage I non-small cell lung cancer",
"data-origination": "SUBMITTED"
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