Agencourt ampure xp reagent beads

At the end of the 8th week, fresh feces was directly collected into sterilized 2 mL Eppendorf tubes and then stored in a −80 °C refrigerator until being analyzed. The fecal microbiotic distribution was analyzed with a 16s RNA Next Generation Sequencing (NGS) system, using the Greengenes database for classification. Agencourt AMPure XP Reagent beads (Beckman Coulter, Brea, CA, USA) were used to purify the amplified DNA. A quantitative (q)PCR (KAPA SYBR FAST qPCR Master Mix) was used to quantify each library in the Roche LightCycler 480 system (Roche, Basel, Switzerland). Then, each sample was diluted to 4 nM for the Illumina MiSeq NGS system (Illumina, Foster, CA, USA). The QIIME package tool was used for bioinformatics microbiome analyses, including operational taxonomic unit (OTU) assignment, phylogenetic reconstruction, diversity analyses, and visualization. OTUs were assigned for each sequence based on 97% similarity.

Samples positive for E. falciformis and E. vermiformis from Mus musculus and Eimeria spp. from Apodemus with indistinguishable 18S and COI sequences were used for a multimarker amplification using the microfluidics PCR system Fluidigm Access Array 48 x 48 (Fluidigm, San Francisco, California, USA). We used target‐specific primers (Data S3) that were designed based on the genome of E. falciformis (Heitlinger et al., 2014) to amplify exons of nuclear genes (Data S4) and coding and noncoding regions from the apicoplast genome (Data S5). Library preparation was performed according to the protocol Access Array Barcode Library for Illumina Sequencers (single direction indexing) as described by the manufacturer (Fluidigm, San Francisco, California, USA). The library was purified using Agencourt AMPure XP Reagent beads (Beckman Coulter Life Sciences, Krefeld, Germany). Quality and integrity of the library was confirmed using the Agilent 2200 Tape Station with D1000 ScreenTapes (Agilent Technologies). Sequences were generated at the Berlin Center for Genomics in Biodiversity Research (BeGenDiv) on the Illumina MiSeq platform (Illumina) in two runs, one using “v3 chemistry” with 600 cycles, the other “v2 chemistry” with 500 cycles. All sequencing raw data can be accessed through the BioProject PRJNA548431 in the NCBI Short Read Archive (SRA).

The fecal microbiotic composition was analyzed using a 16S ribosomal (r)RNA Next Generation Sequencing (NGS) system. Fresh feces were collected into sterilized 2-mL Eppendorf tubes and stored at −80 °C for analysis. The amplified fecal DNA was purified with Agencourt AMPure XP Reagent beads (Beckman Coulter, Brea, CA, USA). A qPCR (KAPA SYBR FAST qPCR Master Mix) was used to quantify each library with the Roche LightCycler 480 system, and then pooled equally to 4 nM for the Illumina MiSeq NGS system (illumina, San Diego, CA, USA). More than 80,000 reads with paired-end sequencing (>250 bp*2) were generated, and the QIIME2 workflow [60 (link)] classified organisms from the amplicons using the GreenGenes taxonomy database. MicrobiomeAnalyst [61 (link)] was used to perform statistical and visual analyses of the microbiome data.