Supplementary file 1 (S1)

Library preparation and DNA sequencing

We followed the detailed description provided by Jervis-Bardyet al. (2015) for 16S rRNA library preparation along with the Illumina 16sMetagenomic Library Prep Guide, 15044223-b( As the preferable DNA quantities recommended by Illumina were higher than our DNA yields, we modified these protocols to accommodate our need for low DNA concentrations. The primer pair we used in our study is derived from Klindworth et al. (2013) and targeted the hypervariable regions V3 (S-D-Bact-0341-b-S-17: 5’-CCTACGGGNGGCWGCAG-3’) and V4 (S-D-Bact-0785-a-A-21: 5’-GACTACHVGGGTATCTAATCC-3’) of the 16S rRNA gene. Primers were connected to Illumina overhang adapter sequences as follows:

forward: 5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3’,

reverse: 5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3’.

The first amplicon PCR was performed with 12.5 µL KAPA HiFiHotStartReadyMix (KAPA Biosystems, MA, USA), 5 µL DNA, 2.5 µL of each primer (2 µM) and 5 µL of aqua dest. totaling to a volume of 25 µL.

PCR was performed in a TProfessional standard Thermocycler (Biometra GmbH, Göttingen, Germany) using the following program: Initial enzyme activation at 95 °C for 3 min, then 30 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 30 s and 5 min of 72 °C for final extension with subsequent cooling at 4 °C.

For the PCR clean-up, amplicon PCR plate was centrifuged at 1,000 x g for 1 min and magnetic AMPure beads (AgencourtAMPure XP Kit, Beckman Coulter, Inc., Brea, CA, USA) were vortexed for 30 s. Then, 20 µL of ofAMPure beads solution was added to each sample and the entire volume was pipetted up and down at least 10 times. The plate was sealed, incubated at room temperature for 5 min and rested afterwards for 4 min on a magnetic stand (Invitrogen™ Ambion™ Magnetic Stand-96, life technologies™, Carlsbad, CA, USA). Supernatant was removed by pipetting. Two ethanol washes were performed consecutively: Beads were rinsed in each well using 200 µL of 80% ethanol and incubated on the magnetic stand for 30 s, supernatant was removed carefully. After a thorough removal of remaining ethanol by pipetting, beads dried for 10 min on the magnetic stand. Preliminary investigations showed that PCR product yield is close to the lower limit of recommended amounts for sequencing. Samples were therefore handled with special care and reserve amounts were reduced to a minimum. We added only 14 µL of 10 mMTris buffer (pH 8.5) following the drying step. Beads were resuspended by pipetting up and down 10 times at least, incubation at room temperature took 2 min. The plate rested another 3 min on the magnetic stand before we transferred 13 µL of supernatant to the new plate.

The size of the PCR products was verified on an Agilent 2100 Bioanalyzer system using a DNA 1000 Chip (Agilent Technologies, Palo Alto, California, USA). Results of the bioanalyzer showed amplicon lengths (~550 bp) as expected but low amounts in 22 samples. Therefore, we repeated the above described PCR for 22 samples to enhance amplicon yield. The PCR products of the identical samples were pooled and once more cleaned. Another bioanalyzer run showed sufficient amounts for the next preparation step.

The Nextera XT Index Kit (lllumina, Inc., San Diego, CA, USA) with dual 8 basemultiplex identifiers (MIDs) was used to introduce unique barcodes and sequencing adapters to each sample and thus allow for multiplexing. The MIDswere located at either end of the amplicon and were chosen according to the manufacturer’s recommendations.

PCR to introduce the unique barcodes to the 16S amplicons (index PCR) was performed in a TProfessional Thermocyclerusing 25 µL KAPA HiFiHotStartReadyMix, 10 µL of cleaned PCR product, 4 µL of each i5 and i7 index (Illumina) and 7 µL aqua dest. The PCR programme began with initial heat activation of the polymerase at 95 °C for 3 min, followed by 8 cycles of 95 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s and a 5 min final extension at 72 °C, and then holding at 4 °C.

Second PCR clean-up was performed, starting with centrifuging the plate at 280 x g for 1 min at room temperature and vortexing the AMPure beads for 30 s. 56 µL of bead solution was added to each well and pipette mixed thoroughly 15 times. Beads settled for 4 min on the magnetic stand and supernatant was removed subsequently. Two washing steps were performed as described above. 27.4 µL of Trisbuffer (pH 8.5) were pipetted to each well and mixed by pipetting up and down 10 times. After 2 min incubation, index PCR plate rested for 3 min on the magnetic stand. In two steps, 25 µL of the supernatant was transferred to a new plate (20 µL and 5 µL to avoid uptake of beads).

The size of the libraries (expected ~630 bp) was verified on anAgilent 2100 bioanalyzersystem using a DNA 1000 Chip (Agilent Technologies, Palo Alto, California, USA). Library concentrations were determined using the Quant-iT™ PicoGreen dsDNA Assay Kit (Invitrogen, Molecular Probes, Eugene, OR, USA) on a TECAN Infinite Reader M200 instrument. Finally, the produced libraries were pooled in equal concentrations into one final library, which wassequenced on an Illumina MiSeq system (Illumina, Inc., San Diego, CA, USA) in paired-end mode (2 x 300 sequencing cycles) at the CeBiTec,Bielefeld University, using the MiSeq Reagent Kit v3 (Illumina, Inc., San Diego, CA, USA) following the manufacturer’s manual. The indexed 16S amplicon pools (75%) were sequenced together with a whole genome shotgun library (25%) on a complete MiSeq run. The addition of a genomic library helps to generate better sequencing results, as it increases the diversity during sequencing. In contrast to 16S amplicon libraries, the whole genome shotgun library generates much more diverse signals than the 16S amplicons and thus increases the performance.