L. reuteri MM4-1A ATCC PTA-6475 aliquot 13
DNA was prepared from a frozen aliquot of Lactobacillus reuteri MM4-1A ATCC PTA-6475 using the MoBio/QiaAmp PowerFecal DNA kit according to manufacturers instructions (Qiagen-USA, Germantown, MD). 16S rDNA libraries were prepared essentially according to Illuminas 16S Metagenomic Sequencing Library Preparation reference guide (part# 15044223 B; https://support.illumina.com/downloads/16s_metagenomic_sequencing_library_preparation.html).  V3-V4 region amplicon primers (Klindworth et al., Nucleic Acids Res 2013) were modified to include a heterogeneity spacer containing 0 to 3 random nucleotides between the 16S sequence and the adaptor sequence, in order to create more sequence diversity and eliminate the need for PhiX spike-in (Fadrosh et al., Microbiome 2014).  Libraries were pooled and sequenced using Illumina MiSeq v3 600 cycle reagents, according to manufacturers instructions.