title: T1 sample 4
design: To analyze the effect of AMC treatment on the microbial gut composition, the 16S rRNA encoding operon was sequenced by Nanopore Oxford Technology (MiniION). From frozen stools (250 mg), DNA was extracted and purified using the DNA mini Stool. Similarly, DNA was extracted from 250 L of a predefined bacterial community (Fecal Reference with TruMatrix Technology, ZymoBIOMICS, Germany). The V1-V9 region of the 16S rRNA operon was amplified using 10 ng of DNA extracts and 27F and 1492R primers. The amplification was obtained using the LongAmp Hot Start Taq 2X Master Mix (New England Biolabs, France) with the following cycling steps: initial denaturation (1 at 95C) ; followed by 25 cycles including denaturation (20 at 95C), annealing (30 at 55C), extension (2 at 65C). The amplification was concluded by a final extension (5 at 65C). The PCR products were dosed using the Qubit 4 fluorometer and equal amounts of amplicons per sample were used for libraries preparation using the Native Barcoding Kit 24 V14 (SQK-16S114.24) and following the manufacturers instructions (Oxford Nanopore Technologies, UK). Sequencing was performed on an Mk1B MinIon sequencer using an R10.4.1 flow cell.