Miseq instrument

Library preparation was performed with the Archer™ FusionPlex™ Heme Panel v1 with Archer™ Universal RNA Fusion Detection v1 for the Illumina Platform according to the protocols described by the manufacturer (ArcherDX, Boulder, Colorado) (Fig. 1). 200 ng RNA was used as input material. Libraries were purified using Agencourt AMPure Beads on a Life Technologies™ DynaMag™ and quantified with the KAPA Biosystem Library Quantification Kit (Illumina, San Diego, California). Libraries were sequenced by combining four samples, at a concentration of 18pM, using the sequencing kit version 2 and the MiSeq instrument (Illumina, San Diego, California). 10% PhiX was used. Given the size of our clinical laboratory, simultaneous runs of four samples would meet the need to routinely perform the analysis once a week. For the validation cohort, Archer™ FusionPlex™ Heme Panel v2 (ArcherDX, Boulder, Colorado) was used and samples were sequenced in batches of six, using the sequencing kit version 3 and the MiSeq instrument (Illumina, San Diego, California). The Heme Panel v2 was used due to the fact that the v1 panel was no longer commercially available, however, the targets examined are included in both versions.

The nested barcode HCV HVR1 positive PCR products were cleaned using AMPure XP beads (Beckman-Coulter), followed by an Index PCR using indexes and adapters, as required for NGS and demultiplexing. The products of the index PCR were purified using AMPure XP beads and quantified on Tape station instrument (Agilent, California, USA) according to manufacturer’s instructions. Normalization was done by taking the appropriate volume of each fragment and mixing together to create an equimolar pooled library of all positive specimens (n = 25). The pooled library size was checked after purification on 4150 Tape station system (Agilent Inc., USA) followed by the dilution of library to 3nmol/l for the MiSeq sequencing procedure using v3 chemistry on Illumina MiSeq instrument and sequenced on MiSeq instrument (Illumina Inc, USA).

16S rRNA V3-V4 amplicon sequencing library preparation and Illumina MiSeq sequencing were conducted at GENEWIZ, Inc (South Plainfield, NJ, United States). Briefly, microbial genomic DNA for each sample was normalized to 20 ng/μl using a Qubit 2.0 Fluorometer (Invitrogen) and was used to generate amplicons using a MetaVx™ Library Preparation kit (GENEWIZ). The V3-V4 region of the 16S rRNA gene was amplified using the forward and reverse primers (341 Forward: CCTACGGRRBGCASCAGKVRVGAAT, 806 Reverse: GGACTACNVGGGTWTCTAATCC). Second limited-cycle amplification was performed to add multiplexing indices (barcode) and Illumina sequencing adapters. DNA libraries were validated by Agilent 2100 Bioanalyzer (Agilent Technologies) and quantified by Qubit 2.0 Fluorometer (Invitrogen). DNA libraries were multiplexed and loaded on an Illumina MiSeq instrument according to manufacturer’s instructions (Illumina). Sequencing was performed using the 2×250 paired-end configuration, and image analysis and base calling were conducted by the MiSeq Control Software embedded in the Illumina MiSeq instrument.

Respiratory samples for all 127 patients, either nasopharyngeal swabs, oropharyngeal swabs, or tracheal aspirates, were collected and centrifuged for 15 min at 4800×g, and the pellet was used for DNA extraction. DNA was extracted using the QIAmp Cador Pathogen Mini Kit extraction (Qiagen N.V., Hilden, Germany) according to the manufacturer´s instructions. V3–V4 16S rRNA region was amplified by PCR using the primers reported by Klindworth et al.^{54 (link)} (for more information see Supplementary Material S1). Library preparation was done according to the Illumina 16S metagenomic sequencing protocol with few modifications. Briefly, 16S amplicons were purified with the DNA clean & concentrator kit (Zymo Research, Irvine Cal., USA). Dual indices and Illumina sequencing adapters were attached in a second PCR step using Nextera XT Index Kit V2 (Illumina, San Diego Cal., USA). Finally, amplicons were purified, pooled in equimolar concentrations, and sequenced in a MiSeq Illumina instrument generating paired-end reads of 250 bp.

We applied strict laboratory protocols to ensure the validity of our molecular data (Supplementary Methods). For each sediment layer, two DNA extractions were performed on 0.5 g of wet sediment using the NucleoSpin® soil kit, according to the manufacturer instructions (Macherey-Nagel, Düren, Germany). The V7 region of the 18S rRNA gene was PCR amplified on a 260-bp-long fragment, from c.a. 25 ng of environmental DNA extracted from each sample and using the general eukaryotic primers 960 F (5′-GGCTTAATTTGACTCAACRCG-3′)^{63 (link)} and NSR1438 (5′-GGGCATCACAGACCTGTTAT-3′)^{64 (link)}. The choice of the barcode region has been validated as reported by Capo et al.^{17 (link),18 (link)}. Details about the preparation of purified amplicons are given in Supplementary Methods.
The purified amplicons for each sample were pooled at equimolar concentrations and sent to GeT-PlaGe (Plateforme Génomique 31326 CASTANET-TOLOSAN Cedex) for library preparation and paired-end (2 × 250 bp) sequencing on a MiSeq Illumina instrument (San Diego, CA, USA).

DNA directly extracted from fecal samples was used to assess the microbiota by the amplification of the V3-V4 region of the 16S rRNA gene using the primers and protocols described by Klindworth et al.^{59 (link)}. PCR amplicons were cleaned using Agencourt AMPure kit (Beckman Coulter, Milan, Italy) and the resulting products were tagged by using the Nextera XT Index Kit (Illumina Inc. San Diego. CA) according to the manufacturer’s instructions. After the 2nd purification step, amplicons products were quantified using a QUBIT dsDNA Assay kit (Life Technologies). Subsequently, equal amounts of amplicons from different samples were pooled. The pooled sample was run on an Experion workstation (Biorad, Milan, Italy) for quality analysis prior to sequencing. The sample pool (4 nM) was denatured with 0.2 N NaOH, diluted to 12 pM, and combined with 20% (vol/vol) denatured 12 pM PhiX, prepared according to Illumina guidelines. The sequencing was performed with a MiSeq Illumina instrument (Illumina) with V3 chemistry and generated 250 bp paired-end reads according to the manufacturer’s instructions.