DNA amplification, library preparation and sequencing
To investigate the gut microbiomes of bats (n=8) and birds (n=10), we followed the earth microbiome protocol (Caporaso et al. 2010). Moreover, we added four samples consisting of a ZymoBIOMICS microbial community standard D6300 (Zymo Research Europe, Freiburg, Germany). These were used as positive controls for microbiome amplification and allowed us to examine whether the clamps had any effect over the yield of a normal sample depleted of chloroplast and mitochondria. The extracted DNA was amplified with the universal bacterial primers 515F (5’-GTGCCAGCMGCCGCGGTAA-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT-3’). We used a two-step amplification process following the amplicon tagging scheme of Fluidigm (Access Array System™ for Illumina Sequencing Systems, ©Fluidigm, San Francisco, USA). In the first step, we amplified a 291 bp fragment of the hypervariable V4 region of the 16S rRNA gene by using tagged (CS) target specific (TS) primers: CS1-NNNN-TS-515F and CS2-TS-806R. We added four random bases to our forward primers to facilitate cluster identification during the first cycles on the Illumina Miseq System. In the second step, the tags (CS1 and CS2) were used to add a sample-specific 10 bp barcode and the Illumina system adapters.
The initial 15 μl PCR volume contained 1.5 μl (5-15 ng) extracted DNA, 7.5 μl DNA polymerase AmpliTaq Gold™ 360 Master Mix (Applied Biosystems, Darmstadt, Germany), 1.5 μl (0.2 μM) primers and 4.5 μl sterile water. The PCR protocol consisted of an initial activation step at 95°C for 10 minutes, followed by 30 cycles at 95°C for 30 seconds, 60°C for 30 seconds and 72°C for 45 seconds, and a final elongation at 72°C for 10 minutes. When clamps where implemented, the water volume was reduced to 1.5 μl; the 1.5 μl from each clamp (mPNA and either cpPNA or pPNA) was added to this first step to give a final concentration of 1 μM.
The modified PCR protocol included a step in order to allow the binding of the PNA to the target sequences (Fig 3). For the second barcoding PCR (20 μl), we used 3 μl initial PCR product, 10 μl AmpliTaq Gold™ 360 Master Mix, 4 μl (0.4 μM) barcode primers (Fluidigm) and 3 μl sterile water. PCR conditions were the same as before, but only 10 cycles were performed.
We used the NucleoMag® NGS Clean-Up and Size Select Kit (Macherey-Nagel, Düren, Germany) on a GeneTheatre® (Analytik Jena, Jena, Germany) to clean the PCR products according to the manufacturer’s guidelines. We assessed the quality of the amplicons by using the QIAxcel Advanced System® (QIAGEN, Hilden, Germany) and then proceeded to quantify the DNA concentration by using the PicoGreen QuantiFluor® dsDNA System (Promega, Madison, USA) on a TECAN Infinite F200 PRO® plate reader (Tecan, Männedorf, Switzerland). We normalized the library to include 20 ng DNA from each sample. Finally, we diluted the library to 3 nM for sequencing. The library was spiked with 5% PhiX sequencing control V3 (Illumina, San Diego, USA). Paired-end sequencing of the amplicons was performed as recommended by Illumina (Miseq Reagent Kit v2 – Reagent Preparation Guide) and loaded at a final library concentration of 6 pM. Paired-end sequencing was performed over 2 x 250 cycles.