V3-V4 of 16S rRNA sequencing
Total DNA was isolated directly from the pla-ra samples (200 mg pla-ra/sample) using a QIAamp DNA stool mini kit (Qiagen, Hilden, Germany) as recommended by the manufacturers. DNA concentration and purity were monitored on 1% agarose gels. Each DNA sample was then diluted to 1 ng/l 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 products (ranging between 400-450 bp in length) were quantified, qualified and chosen for library preparation. The libraries were generated using an Ion Plus Fragment Library Kit (Thermo Fisher Scientific, Waltham, MA) and quantified using Qubit and qPCR, then sequenced on an IonS5TMXL instrument. Single-end reads were generated.