fastq files - Experiment 1

<b>Sample collection</b>We sampled black-capped chickadee feces in December 2020 in the municipality of Chertsey, Quebec, Canada (46°08'24.4"N, 73°49'58.7"W). We collected 150 fresh bird fecal samples by placing a plastic canvas below a feeder exclusively used by black-capped chickadees during the sample collection period. We collected the feces on cold days so that the feces froze immediately after defecation. The feces did not spend more than three hours on the canvas (normally less than an hour). Between May and June 2021, we sampled 13 feces of blue tit nestlings aged 15 days, nesting in artificial nest boxes northwest of Montpellier, France (43°39'56"N, 3°40'1"E). We stored all the feces in 1 mL of 95% ethanol at -20 C. Ethanol is commonly used to prevent microorganism proliferation and to preserve the original microbiota composition in samples (Marotz et al. 2021). For each species, we pooled and homogenized all the feces by stirring with a sterilized metal spatula in a sterilized plate for five to ten minutes. We split the pooled samples into 300 replicates for black-capped chickadees and 25 replicates for blue tits. This step was performed to reduce variability in bacterial composition among samples and facilitate the comparisons of the kits and methods by creating technical replicates from the same homogenized pool of fecal samples.<b>Experimental design</b>For the black-capped chickadee feces samples, we compared five commercial DNA extraction kits: 1) MagMAX Microbiome Ultra Nucleic Acid Isolation Kit by Fisher Scientific (MagMAX), 2) Quick-DNA Fecal/Soil Microbe Microprep Kit by Zymo Research (QuickDNA), 3) DNeasy PowerSoil Kit by Qiagen (PowerSoil), 4) Stool DNA Isolation Kit by Norgen Biotek (StoolNorgen), and 5) PureLink Microbiome DNA Purification Kit by Invitrogen (PureLink).For each kit, we compared two solutions (sterile water or PBS) for rinsing samples that had been stored in 95% ethanol. For the PowerSoil, StoolNorgen, and PureLink kits, we compared two elution methods (elute once or twice). We tested 16 different conditions ([3 kits x 2 elution methods] + [2 kits x 1 elution method] x 2 rinsing solutions). We used 15 samples and a negative control for each condition. As a negative control, we performed all extraction steps without a sample starting at the bead beating step. For the blue tit samples, we compared the same five extraction kits used for black-capped chickadees, but we used only one preparation method: samples were rinsed twice with PBS and were eluted once. We used five samples and a negative control for each kit.<b>DNA extraction</b>To rinse the samples, we centrifuged the tubes containing the samples for 10 min at 10,000 rpm, removed the ethanol with a micropipette, added 1 mL of ether sterile water or PBS, vortexed the tubes for 3 sec, centrifuged again for 10 min at 10,000 rpm and repeated these steps a second time. We followed manufacturer protocols for DNA extraction except that we increased the bead beating time to 20 min for each kit.For black-capped chickadee samples extracted with PowerSoil, StoolNorgen, and PureLink, we performed a first elution with 60 L of elution solution and a second with 45 L of the first eluate. This resulted in 15 L of DNA product that passed once through the column filter and 45 L that passed twice. For all blue tit samples and for black-capped chickadee samples extracted with MagMAX and QuickDNA, we eluted once with 60 L of elution solution. We quantified DNA concentration in all samples using a Qubit 3.0 high sensitivity fluorometer.<b>DNA library preparation and sequencing</b>For the remaining steps, we randomly selected five samples and an extraction negative control per condition (samples extracted with the same kit, rinsed with the same solution, and eluted the same number of time) for both species. For each sample, we performed a PCR amplification of the V5-V6 regions of the bacterial 16S rRNA gene using host DNA-excluding 799F/1115R primers (Chelius and Triplett 2001). The PCR was performed in a 25 μL mixture containing 1 μL of DNA extract, 0.2 μM of each primer, 0.5 U of Phusion Hot Start II High-Fidelity DNA Polymerase (Thermo Fisher Scientific, Waltham, Massachusetts, United States), 1X of Phusion HF Buffer, 0.2 mM of dNTPs, 3% DMSO and following this programme: initial denaturation at 98 °C for 30 s, 35 cycles of denaturation at 98 °C for 15 s, annealing at 64 °C for 30 s and elongation at 72 °C for 30 s and an extension step of 72 °C for 10 min. For PCR negative controls, we replaced DNA by sterile water and for PCR positive controls we used ZymoBIOMICS Microbial Community DNA Standard (Zymo Research, Irvine, California, United States). We visualized all PCR products on 2% agarose gel, and we normalized them using Just-a-plate 96 PCR Purification and Normalization kit following the manufacturer’s protocol (Charm Biotech).The black-capped chickadee samples were pooled together to form a sequencing library and the blue tit samples from MagMAX, PowerSoil, and PureLink were also pooled together in another library. Blue tit samples extracted with Quick DNA and StoolNorgen kits did not amplify, and we thus chose not to sequence them. We sequenced samples from PowerSoil even if they did not amplify because of its wide used in the literature (Cao et al. 2020; Grond et al. 2019; Kreisinger et al. 2017). The pooled samples were purified with a ratio of 0.75 of NucleoMag using the manufacturer protocol (Macherey-Nagel, Germany). Quality control of the libraries was assessed as follows: libraries were quantified using the Qubit dsDNA HS Assay Kit (Invitrogen, Carlsbad, California, United States) and the NEBNext Library Quant Kit for Illumina (New England BioLabs, Ipswich, Massachusetts, United States) and the average fragment size was determined using a Bioanalyzer instrument (Agilent Technologies, Santa Clara, California, United States). Before sequencing, the PhiX control library (Illumina, San Diego, California, United States) was spiked into the amplicon pool to improve sequence yield. Sequencing was performed on an Illumina Miseq at the UQAM CERMO-FC Genomics Platform using the MiSeq reagent kit v3 (2 x 300 cycles; Illumina). The black-capped chickadee samples were sequenced in one run and the blue tit samples in another run.