Phix sequencing control

The DNA extracted from six infant feces samples per group served as a template to prepare the 16S rDNA library following Illumina’s 16S Metagenomic Sequencing Library Preparation guide. These infants were born by vaginal delivery, and their mothers had healthy BMI in the three times mentioned above.
Briefly, PCR amplification of the V3 and V4 regions of the 16S rRNA gene (approximately 460 bp fragment) was conducted with the Phusion® High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA, USA). Thermal cycling conditions were performed according to the manufacturer´s protocol in a Veriti 96-Well Thermal Cycler (Applied Biosystems, Foster City, CA, USA). For each biological sample, three technical PCR replicates were amplified. Positive amplification was verified by 1.5% agarose gel electrophoresis. Pooled PCR replicates were purified with Agencourt AMPure XP beads (Beckman Coulter Life Sciences, Indianapolis, IN). Library quantification and normalization were conducted with a Qubit® dsDNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA) in a Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA). Paired-end sequencing (2x300 pb) was performed with a MiSeq Reagent Kit v3 (Illumina, San Diego, CA, USA) at a final loading concentration of 8 pM in a MiSeq System (Illumina). PhiX sequencing control (Illumina) represented 30% of the pooled library at 8 pM.

Pooled quantified library was processed for MiSeq sequencing by diluting to 4 nM and then denatured by mixing 1:1 with 0.2 N NaOH, for a final concentration of 2 nM of DNA and 0.1 N NaOH. Then, the library was mixed at a 1:1 ratio with 2 nM denatured PhiX Sequencing Control (Illumina). Three primers were used for sequencing: read 1 (5’-TATGGTAATTGTGTGCCAGCMGCCGCGGTAA-3’), read 2 (5’-AGTCAGTCAGCCGGACTACHVGGGTWTCTAAT-3’) and index (5’-ATTAGAWACCCBDGTAGTCCGG CTGACTGACT-3’). We used the MiSeq kit V2 for 500 cycles (Illumina) for sequencing, following the manufacturer’s instructions.

Primers (Sigma) were designed to amplify the regions of the genes that contained hotspot mutation and were built with different 5′-adapter region. To prepare the next-generation sequencing (NGS) library, we carried out two consecutive rounds of PCR, both of which used Phusion Hot Start II High-Fidelity DNA Polymerase (Thermo Fisher). In the first round, primers recognized and amplified the region(s) of interest, and in the second one, PCR products from first round were diluted 1:100 and then amplified with Index primers (IDT) that barcoded each sample for subsequent univocal identification. After amplification, Wizard^® SV Gel and PCR Clean-Up System (Promega) were used to purify the fragments, following the manufacturer’s instructions. Finally, all samples were diluted to a final concentration of 2 nM, to create the library. Phix sequencing Control (Illumina) was added and the library was finally denatured and loaded into the cartridge (Illumina).
Sequencing data were exported as BAM and VCF files and analyzed using the IGV software and the VariantStudio software (Illumina), respectively (Supplementary Data Sheet 1). After quality check controls and filtering, reads and variants distributed in target regions were analyzed and annotated.