2.3 Bioinformatics, sequence analysis and statistical analysis
In
order to obtain more reliable and high-quality sequencing results (valid
reads), the following pre-procedures were performed on the raw reads
from the Illumina MiSeq platform: raw reads were
demultiplexed,
quality-filtered by FASTP (version 0.19.6; Chen, Zhou, Chen, & Gu,
2018) and merged by FLASH (version 1.2.11; Magoc & Salzberg, 2011), and
high-quality reads were clustered as an operational taxonomic unit (OTU)
by UPARSE
(version
7.0) when the sets of sequences shared at least 97% identity (Edgar,
2013), and chimeric sequences were identified and removed. All OTUs with
totaling reads more than 50 were used. The taxonomy of each OTU
representative sequence was analyzed by RDP Classifier (version 2.11;
Wang, Garrity, Tiedje, & Cole, 2007) against the Silva 16S rRNA
database
(version 138) using confidence
threshold of 70% (Quast et al., 2013).
Mothur software (version 1.30.2) was employed to calculate α-diversity
including Sobs, Chao1, Shannon, Simpson, and Coverage, and Student’st -test was performed to compare α-diversity estimates andP -value less than 0.05 was considered statistically significant.
β-diversity analysis was performed
and visualized with principal coordinates analysis (PCoA) were
determined by Bray-Curtis distances, based on OTU compositions and
Adonis test (with 999 permutations) was conducted to show
differentiation in microbial structures of different sexes.
Taxa abundances in two sexes at the phylum and genus levels were
compared by
Wilcoxon
rank-sum test and a two-tailed P -value less than 0.05 was
considered significant (with bootstrap values 95%). The different
biomarkers associated with sex were characterized by linear discriminant
analysis (LDA) effect size (LEfSe) (Segata et al., 2011). Microbial
functions were predicted by using phylogenetic investigation of
communities by reconstruction of unobserved states 2
(PICRUSt2)
based on high-quality sequences, and an
Independent
sample t -test was performed to measure whether the difference
between the two sexes is significant (SPSS, version 26.0), and the
diagrams were finished by Origin (version
2019).