16S rRNA bioinformatic analysis
The 16S rRNA analysis workflow is shown in Figure 2. The results from the next-generation sequencing were in the FAST5 format. The FastQ files were generated from the results in the FAST5 format by using the Guppy program in MinKNOW (Oxford Nanopore Technologies, UK), in which real-time quality scores of more than 7 were filtered and demultiplexed. The FastQ passing quality was aligned to reference sequences in the NCBI database by using the FASTQ 16S workflow (version 2022.01.07), and the inclusion criteria were a minimum BLAST e-value of 0.01, minimum coverage of more than 30%, and a minimum identity of more than 77%. The data output files were generated as CSV files. Then, the percentage prevalence of each species and genus were calculated, and species and genus were filtered at a level of 0.5% for investigation of relative abundance.
Comparative taxonomy was performed to investigate the relative abundance of species between neonates delivered vaginally and those delivered by cesarean delivery.