Ion chef system

The extracted total RNA or miRNA was enriched for small RNA using a Magnetic Bead Cleanup Module (Thermo Fisher, Waltham, MA, USA), as per the manufacturer’s instruction. The quality and quantity of the enriched small RNAs were assessed by the Agilent 2100 Bioanalyzer instrument with the Agilent Small RNA Kit (Agilent Technologies, Santa Clara, CA, USA). Then, the small RNAs were reverse-transcribed using the Invitrogen™ SuperScript™ VILO™ cDNA Synthesis Kit (Thermo Fisher, Waltham, MA, USA). Small RNA libraries for sequencing were generated using the Ion Total RNA-Seq Kit v2 (Thermo Fisher, Waltham, MA, USA) with the Ion Xpress Barcode Adapters Kit (Thermo Fisher, Waltham, MA, USA) for multiplexing samples. Barcoded libraries were amplified using an emulsion polymerase chain reaction on Ion Sphere particles, and sequencing was performed on an Ion Chef System and an Ion Proton Sequencer (Life Technologies, Carlsbad, CA, USA) using an Ion PI Hi-Q Chef Kit (Life Technologies, Carlsbad, CA, USA). Sequenced reads were trimmed, their quality assessed by a program called FastQC, aligned to the reference genome (hg19) and then counted for miRNA using the SmallRNA Analysis program on the Torrent Suite Software (Thermo Fisher, Waltham, MA, USA).

Next-generation sequencing (NGS) was performed as previously described [4 (link), 7 (link)] by Fujita and Damiati in 2017 and 2016, respectively. In brief, 100 ng of DNA was amplified for genomic library preparation using the exome enrichment kit (Ion AmpliSeq™, Life Technologies, USA) in order to sequence the key exonic regions (> 97% of CCDSs) of the genome. Ion Chef™ System (Life Technologies, USA) was used for template preparation and enrichment using Ion 540™ Kit – Chef (Life Technologies, USA). The same automated platform was used for loading Ion 540™ Chips with template-positive Ion Sphere™ Particles. Exome sequencing was performed on Ion S5™ XL Sequencer (Life Technologies, USA) with the loaded chips. Data analysis was done by Torrent Suite™ Software (v 5.2.2; Life Technologies, USA). Coverage analysis was performed using the Coverage Analysis plug-in (v5.2.0.9). Variant Caller plug-in (v5.2.0.34) was used for mutation/variant detection against the reference genome (hg19).

RNA from plasma (400 μl) was extracted using the Qiagen miRNeasy mini kit with the use of TRIzol-LS (ThermoFisher) for organic phase separation and was analysed by an Agilent 2100 Bioanalyzer using Small RNA Chips (Agilent Technologies). Small RNA libraries were constructed from total isolated RNA using the Ion Total RNA-Seq Kit V2 (Life Technologies, Australia) and ligated to adapters containing a unique index barcode according to the manufacturers’ protocol. The yield and size distribution of the small RNA libraries were assessed using the Agilent 2100 Bioanalyzer instrument with the DNA 1000 chip (Agilent Technologies). Equally pooled libraries were prepared for deep sequencing using the Ion Chef system (Life Technologies) and sequenced on the Ion Torrent Ion S5 using Ion 540 chips (Life Technologies). Pre-processing of reads, removal of adapters and barcodes were performed using the Torrent Suite (v.5.8.0). Sequences were analyzed for quality control (FASTQC), aligned to the Human genome (HG19) using the Torrent Suite, and files transferred to Partek Genomic Suite and Flow (Partek Incorporated, Singapore) for mapping against miRBase V.20 and normalization to reads per million reads (RPM). miRNAs identified with at least 30 RPM were used for further analysis on Partek Genomic suite which included statistical analysis and hierarchical clustering.

The microbiota of the 61 BAL samples included in the study was characterized by high throughput sequencing of the 16S rRNA gene. For this purpose, the 16S Ion Metagenomics kit (Life Technologies) was used, following the manufacturer’s instructions. Briefly, this method includes two separate PCR reactions, amplifying, respectively, V2-4–8 and V3-6, V7-9 regions. Each PCR reaction was carried out on 5 ng of microbial DNA. The thermal profile for both PCR reactions consisted of 1 cycle at 95 °C for 10 min, 30 cycles consisting of 95 °C for 30 s, 58 °C for 30 s, 72 °C for 20 s, and a final extension cycle at 72 °C for 7 min. After amplification, PCR products were purified using the Agencourt AMPure beads (Beckman Coulter Inc, Atlanta, Georgia), eluted in Low TE buffer and quantified by the Qubit dsDNA HS Assay kit (Life Technologies). After amplification, a pool of 100 ng of total DNA for each sample (50 ng from each reaction) was used for library preparation. Libraries were barcoded using Ion Xpress Barcodes Adapters (Life Technologies) and amplified in an emulsion PCR on the Ion Chef system (Life Technologies) according to the manufacturer’s instructions. Sequencing was performed on the Ion S5 System (ThermoFisher) using the Ion 330 Chip kit (Life Technologies). The raw sequences were submitted to NCBI under BioProject accession number PRJNA892871.