Nextera xt kit

Sequencing library was prepared using the Nextera XT Illumina kit, where sequencing adapters and barcodes were added to the samples. The sequencing was carried out at the Laboratory of Medical and Human Genetics (LGHM) at the Federal University of Pará through the Illumina MiSeq platform. PEAR v.0.9.8 (Zhang et al., 2014 (link)) was used to merge the reads. Primers and ambiguous nucleotides were subsequently removed and reads <225 bp were removed as well as reads with total expected error > 0.5. Sequences were clustered into Operational Taxonomic Units (OTUs) based on a dissimilarity of 3% and chimera sequences were removed with UPARSE pipeline (Edgar, 2013 (link)). Taxonomic analysis was performed with USEARCH v.11 comparing with the 16S rRNA database from SILVA v.1.3.2 (Edgar, 2010 (link); Glockner et al., 2017 (link)). Functional properties of the microbial communities were predicted using iVikodak (Nagpal et al., 2019 (link)).
Statistical analysis was performed with Phyloseq, Vegan, and ggplot packages implemented in R Studio v.1.0.136 (McMurdie and Holmes, 2013 (link)). The following alpha-diversity indexes were determined: rarefaction curve, Shannon, Chao1, ACE, and Simpson. Non-Metric Multidimensional Scaling (NMDS) plot was used to compare the bacterial composition of the different seasons.

Stool samples were collected by the patient or nursing staff according to the GUT (DNA Stabilized-frozen Inc., Ottawa, Ontario, Canada) extraction kit. Samples were stored at -80°C till processing. Faecal DNA was extracted using the PowerSoil DNA Extraction Kit (MO BIO Laboratories, Carlsbad, CA, USA), which was then fragmented into 300 bp clone-sized libraries using Nextera-XT Illumina kit (Illumina, Inc. San Diego, CA, USA) and sequenced in an Illumina HiSeq sequencer (Illumina, Inc. San Diego, CA, USA) with a sequencing depth target of 20 million reads.

In addition to 16S rRNA gene sequencing, a subset of 54 samples was evaluated using shotgun fecal metagenomic sequencing. Subjects chosen for shotgun sequencing analysis had fecal samples available for testing at least at months 1 and 4. If available, month 9 from these same subjects was also sequenced. Extracted, non-diluted DNA was fragmented with the Nextera-XT Illumina kit (Illumina, Inc.) following the manufacturer’s instructions. One library of approximately 300 bp clone insert sizes was constructed per sample. Samples were sequenced in an Illumina HiSeq sequencer (Illumina, Inc.).

Genomic DNA was extracted from independently grown colonies on solid rich media (YPD, Yeast extract, Peptone, Dextrose) with DNeasy Blood & Tissue Kit (Qiagen, Hiden, Germany). Libraries were prepared with the Illumina Nextera XT kit (Illumina, San Diego, USA) following the manufacture’s protocol. Libraries and input DNA were quantified with AccuClear Ultra High Sensitivity dsDNA Quantitation Kit (Biotium, Fremont, USA) using the Fusion Optics (SPARK, TECAN, Männedorf, Switzerland). Pooled libraries were sequenced on a single lane of HiSeqX (150PE, Illumina, San Diego, USA) at the Genome Quebec Innovation center (Montréal, Canada). The 74 strains were sequenced with an average genome-wide coverage of ×45(Supplementary Figure 1, Supplementary Data 2). Raw sequences are accessible at NCBI (bio project ID PRJNA479851).