Total DNA was isolated directly from the pla-ra samples (200 mg per sample) using a QIAamp® DNA stool mini kit (Qiagen, Hilden, Germany) as recommended by the manufacturers. Briefly, the pla-ra samples were extracted in optimized buffers in combination with inhibitEX buffer (Qiagen) to remove PCR inhibitors. Proteinase K was subsequently added. DNA from lysates was isolated using a DNA-binding silica-gel membrane column. Remaining impurities were efficiently removed in two wash steps. Amplification-ready DNA was then eluted in low-salt buffer (Qiagen). DNA concentration and purity were monitored using a NanoDrop™ One Spectrophotometer (Thermo Fisher Scientific, Waltham, MA) and electrophoresis through 1% agarose gels. Each DNA sample was then diluted to 1 ng/μl using sterile water.

The V3-V4 regions of the 16S rRNA gene were amplified using universal region-specific primers 341F (5’-CCT AYG GGR BGC ASC AG-3’) and 806R (5’-GGA CTA CNN GGG TAT CTA AT-3’) tagged with sample-identifying barcodes. All PCR reactions were carried out with Phusion® High-Fidelity PCR Master Mix (New England Biolabs, Ipswich, MA). The PCR cycling conditions were as follows: initial denaturation at 98°C for 1 min, followed by 30 cycles at 98°C for 10 s, 50°C for 30 s, and 72°C for 30 s, and a final extension step at 72°C for 5 min. The PCR products (400–450 bp in length) were quantified, qualified and used for library preparation. The libraries were generated using an Ion Plus Fragment Library Kit (Thermo Fisher Scientific) and quantified using Qubit and qPCR, then sequenced on an IonS5TMXL instrument (Thermo Fisher Scientific). Single-end reads were generated.

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