DNA extraction, library building construction, metagenome assembly, and analysis

Total DNA of 10 fourth stage Cx. quinquefasciatus larvae was extracted using the DNeasy Blood & Tissue kit (QIAGEN) and the metagenomic library was prepared using the TruSeq DNA Nano kit. Paired-end sequencing was conducted in a Novaseq platform with a 150 bp read length, generating 32 Gbp in raw reads. Quality check of reads was performed with FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Filtering of Wolbachia reads was computed with Bowtie251 using publicly available Wolbachia from Cx. quinquefasciatus genomes. With the extracted Wolbachia reads the genome was assembled using SPAdes52. The completeness of the genome was checked with BUSCO53 and an alignment with a public available wPip genome was performed using Mauve software54 to check for genomic rearrangements and differences. The phylogenetic reconstruction was performed with GET_PHYLOMARKERS55 using 29 available Wolbachia genomes listed in NCBI genome database (Table S3). This pipeline identifies marker genes and estimates genome phylogenies from the orthologous clusters computed by GET_HOMOLOGUES56. The phylogeny was computed with IQ-TREE57 and the tree was edited with iTOL58.

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