br Tumor stage no br chosen as the

Tumor stage, no
chosen as the representative sequences of corresponding OTUs. Taxo-
nomic assignment of individual datasets were classified against the
Greengenes database version 13.8 using both RDP classifier and
HP infection, no
that were identified as members of Eukarya, Archaea, Mitochondria,
Positive 193
Chloroplasts and Cyanobacteria lineages, were removed. Alpha diversity
Negative 83
was calculated with QIIME software with Python scripts Cell Counting Kit-8 on the se-
Antibiotics use, no
quence similarity at 97% level, including index of observed species,
abundance-based coverage estimator (ACE), Chao1 estimator, Shannon,
Pre-operative chemotherapy, no
Sample collection
Simpson, Evenness and PD whole tree. Sequence coverage was assessed
in mothur by rarefaction curves and Good's coverage [30,31]. Beta di-
versity was measured by unweighted and weighted UniFrac distance
calculated with 10 times of subsampling by QIIME. These distances
were visualized by principal coordinate analysis (PCoA) [32]. Hierarchi-
cal clustering was performed using Spearman's rank correlation coeffi-
cient as a distance measure and a customized script developed in R
version 3.5.1. The output file was further analyzed using Statistical Anal-
ysis of Metagenomic Profiles software package (STAMP) version 2.1.3
BMI: Body mass index; HP, Helicobacter pylori; no, number; PPI, Proton pump inhibi-
tors; SD, standard deviation.
For the predictive functional analyses, PiCRUSt software package
version 1.0.0 was used to identify predicted gene families and associ-
of Medicine, Zhejiang University (China). Informed written consent was ated pathways from inferred metagenomes of taxa of interest identified
obtained from each of the patients before enrollment. from the compositional analyses, which was based on the fact that phy-
logeny and function are closely linked [34]. Predicted functional genes
2.2. Samples collection, DNA isolation, amplicon library construction and were categorized into Clusters of Orthologous Groups (COG) and into
sequencing
Kyoto Encyclopedia of Genes and Genome (KEGG) orthology (KO),
and compared across patient groups using STAMP. Pathways and
After gastrectomy, tissues were rinsed with sterile water, flash fro- enzymes were assigned using KEGG database options built into the
zen in liquid nitrogen, and characterized by staff pathologists. Bacterial pipeline. The pathways that were nonprokaryotic, had fewer than 2 se-
genomic DNA was extracted from tissue samples by using the QIAamp quences in each cohort, or had a difference in mean proportions b 0.1%
DNA Mini Kit (QIAGEN, Hilden, Germany) according to the manufactur- were excluded from analysis. The characterization of microorganismal
er's instructions with minor modifications [22]. Amplicon libraries were features differentiating the gastric microbiota was performed using
constructed with Illumina sequencing-compatible and barcode-indexed the linear discriminant analysis (LDA) effect size (LEfSe) method
bacterial PCR primers 319F/806R, which target the V3-V4 regions of 16S (http://huttenhower.sph.harvard.edu/lefse/), which emphasizes both
rRNA gene [23]. All PCR reactions were performed with KAPA HiFi statistical significance and biological relevance [35]. With a normalized
HotStart ReadyMix using the manufacturer's protocol (KAPA relative abundance matrix, LEfSe uses the Kruskal-Wallis rank sum test
Biosystems) and approximately 50 ng of extracted DNA per reaction. to detect features with significantly different abundances between
Thermocycling conditions were set at 95 °C for 1 min, 55 °C for 1 min, assigned taxa and performs LDA to estimate the effect size of each fea-
then 72 °C for 1 min for 30 cycles, followed by a final extension at 72 ture. A significant alpha at 0.05 and an effect size threshold of 2 were
°C for 5 min. All PCR reactions were performed in 50 μl triplicates and used for all biomarkers discussed in this study.
combined after PCR. The amplicon library was prepared using a Correlation analysis was performed using sparse compositional cor-
TruSeq™ DNA sample preparation kit (Illumina Inc., San Diego, CA, relation (SparCC) algorithm on the complete OTU table collapsed to the
USA). Prior to sequencing, the PCR products were extracted with the genus level, which was introduced by Friedman and Alm and was
MiniElute® Gel Extraction Kit (QIAGEN) and quantified on a NanoDrop known for its robustness to the compositional effects that are influenced
ND-1000 spectrophotometer (Thermo Electron Corporation) and Qubit