Paired end platform

Seventeen DNA samples from pools of ten abdomens of each Anopheles species: An. hyrcanus group (HYR; n = 4 pools), An. nivipes (NIV; n = 5 pools), An. philippinensis (PHI; n = 4 pools), and An. vagus (VAG; n = 4 pools) were used for amplification and sequencing. The universal region-specific primers 341F (5’-CCT AYG GGR BGC ASC AG-3’) and 806R (5’-GGA CTA CNN GGG TAT CTA AT-3’) (NovogeneAIT Genomics Pte. Ltd., Singapore) tagged with sample-identifying barcodes to amplify the V3−V4 regions of the 16S rRNA gene were used. PCR products of approximately 400–450 bp in length were generated. PCR products of the expected size were selected by 2% agarose gel electrophoresis and used for library preparation. At each step of the process, quality control was carried out to maintain the accuracy and reliability of the sequencing data according to the Illumina sequencing system (NovogeneAIT Genomics). The libraries were generated by end-repair, A-tailing, and ligation with Illumina adapters, then assessed using Qubit and qPCR for quantification and a bioanalyzer for size distribution detection. Quantified libraries were sequenced on an Illumina paired-end platform (performed by NovogeneAIT Genomics Pte. Ltd., Singapore) to generate 250 bp paired-end raw reads.

DNA extraction of Cincalok, Tempoyak, and Mandai and subsequent PCR and sequencing were done in a sequencing facility using next-generation sequencing following the amplicon-based approach. Bacterial 16S rRNA gene regions (V4) were amplified using specific primers 515F-806R with the barcode. All PCR reactions were carried out with Phusion® High-Fidelity PCR Master Mix (New England Biolabs, USA). The same volume of 1x loading buffer (containing SYB green) was mixed with PCR products, and electrophoresis was carried out on 2% agarose gel for detection. Samples were sequenced using the Illumina platform. Amplicon was sequenced on the Illumina paired-end platform to generate 250 bp paired-end raw reads (raw PE) and then merged and pretreated to obtain clean tags. The microbial community composition in each sample was studied using Operational Taxonomic Units (OTUs) obtained through clustering with 97% identity on the effective tags of all samples. Subsequently, these OTUs were identified using the SILVA SSU nonredundant database (https://github.com/qiime2/q2-feature-classifier). The taxonomic bar plots were constructed by selecting the top 10 taxa of each sample or group at each taxonomic rank (phylum and genus) to form the distribution histogram of the relative abundance of taxa.

The microbial community composition was analyzed using cell pellets of 4 mL samples taken at the end of a cycle. DNA was extracted from the cell pellets from different time points in the enrichment using the PowerSoil microbial extraction kit (Qiagen Inc., Germany) following manufacturer’s instructions. The DNA-content of the extracts was quantified using a Qubit 4 (Thermo Fisher Scientific, United States) to confirm sufficient DNA was extracted for analysis. The samples were sent to Novogene Ltd. (Hongkong, China) for amplicon sequencing of the V3-V4 region of the 16S-rRNA gene (position 341–806) on an Illumina paired-end platform. After sequencing, the raw reads were quality filtered, chimeric sequences were removed and OTUs were generated on the base of ≥97% identity. Subsequently, the representative sequence for each OTU was annotated using the GreenGene Database. The sequences have been stored in the 4TU research database and can be found under the DOI 10.4121/14394578.

DNA was extracted using the DNeasy UltraClean Microbial Kit (Qiagen, The Netherlands). Approximately 250 mg wet biomass was treated according to the standard protocol except an alternative lysis was implemented. This included a combination of 5 min of heat (65 °C) followed by 5 min of bead-beating for cell disruption on a Mini-Beadbeater-24 (Biospec, USA). After extraction, the DNA was checked for quality by gel electrophoresis and quantified using a Qubit 4 (Thermo Fisher Scientific, USA).
After quality control, samples were sent to Novogene Ltd. (Hong Kong, China) for Amplicon sequencing of the V3–4 region of the 16S-rRNA gene (position 341–806) on an Illumina paired-end platform. After sequencing, the raw reads were quality filtered, chimeric sequences were removed, and operational taxonomic units (OTUs) were generated on the base of ≥ 97% identity. Subsequently, microbial community analysis was performed by Novogene using Mothur & Qiime software (V1.7.0). For phylogenetical determination, a most recent SSURef database from SILVA (http://www.arb-silva.de/) was used.

Total DNA was extracted using (QIAGEN, Hilden, Germany) following the manufacturer’s protocol. Distinct regions (16S V4/16S V3/16S V3-V4) of the 16S rRNA/18S rRNA/ITS genes were amplified using amplicon generation and the specific primers 16SV34, 341F (CCTAYGGGRBGCASCAG), and 806R (GGACTACNNGGGTATCTAAT) with barcodes. All PCRs were carried out with Phusion^® [Editor2] High-Fidelity PCR Master Mix (New England Biolabs). The amplicon was sequenced on an Illumina paired-end platform to generate 250 bp paired-end raw reads (Raw PE), which were merged and pretreated to obtain clean tags. The chimeric sequences in the clean tags were detected and removed to obtain the effective tags that could be used for subsequent analysis. The FASTQ file was merged using the PEAR software package. Operational taxonomic units (OTUs) were defined based on 97% similarity clustering using the UCLUST algorithm. For each representative sequence, the Unite Database (https://unite.ut.ee/ (accessed on 7 February 2022) was used based on the Blast algorithm, which was calculated by QIIME software (Version 1.9.1) (http://qiime.org/scripts/assign_taxonomy.html (accessed on 25 March 2022)) to annotate taxonomic information.
The accepted paired-end, primer-trimmed reads were deposited at the National Center for Biotechnology Information (NCBI) under accession number SUB10994983.

Microbial DNA was extracted from the cervical swab samples by using Genomic DNA Isolation Kit (FairBiotech Corp., Taoyuan, Taiwan). Four hypervariable regions (V3, V4, V5 and V9) of the 16S ribosomal RNA (rRNA) sequence were amplified by PCR. The detail procedure and sequences of the universal primers can be found in the previous study [35 (link),36 (link)]. A total of 10 samples from healthy controls and 23 samples from patients (Table S1) showed clear amplified DNA fragments and were subjected to library preparation using DNA Library Prep Kit (NEB Inc., Ipswich, MA, USA). Amplicon sequencing was conducted with Illumina paired-end platform.