Computational protocol: Draft Genome Sequence of Asaia sp. Strain SF2.1, an Important Member of the Microbiome of Anopheles Mosquitoes

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Protocol publication

[…] Asaia sp. strain SF2.1 is a Gram-negative member of the Alphaproteobacteria, family Acetobacteraceae (). Asaia spp. were first isolated from the nectar of tropical flowers and subsequently from insect midguts (–). The taxonomy of the genus Asaia is in flux and so we are hesitant to assign Asaia sp. SF2.1 to a specific species, although it seems to be most closely related to either Asaia bogorensis or Asaia platycodi (our unpublished data). Asaia sp. SF2.1 was isolated from a laboratory colony of Anopheles stephensi, where it is extremely abundant in the gut, salivary glands, ovaries, and testes of this insect (). Asaia spp. have also been uncovered in Anopheles mosquitoes in the field, especially Anopheles gambiae, the most important vector of malaria in Africa (, , ). Efforts to genetically engineer Asaia sp. SF2.1 are under way in order to provide a platform to deliver antimalarial effector molecules to Anopheles mosquitoes in the field in an effort to block the transmission of malaria, a strategy called paratransgenesis (–). The sequence reported here is the first for the genus.The sequencing and annotation of the genome of Asaia sp. SF2.1 was performed by ACGT, Inc. The standard protocol for the Nextera XT DNA sample preparation kit was used. The purified fragmented DNA was used as a template for a limited cycle PCR using Nextera primers and index adaptors. A second library was prepared using the Nextera mate-pair sample preparation kit.In order to generate clusters of DNA, both libraries were sequenced in a paired-end 2 × 150-bp protocol by MiSeq. The sequence reads passing the Illumina purity filter were demultiplexed. A total of 4,097,892 standard library reads were generated, giving an average coverage of 351× based on the 3.5-Mb genome. To generate additional mate-pair reads, a second MiSeq run was done using the mate-pair library. This run generated 999,241 mate-pair reads (86× coverage).A 93× coverage subset of the small-insert library and the mate-pair library were assembled de novo using ABySS (), Velvet (), and SOAPdenovo2 (). The best Velvet, ABySS, and SOAPdenovo2 contig sets were combined using CISA () to produce an assembly with 51 contigs. The largest contig is 506 kb, the N50 length is 162 kb, and the total assembly length is 3.53 Mb. The G+C content is 59.5%.Annotation of the genome was performed by the NCBI Prokaryotic Genome Annotation Pipeline version 2.0 ( A total of 3,098 genes were predicted using this method, including 3,005 protein-coding genes, 44 pseudogenes, 3 rRNAs, and 45 tRNAs. […]

Pipeline specifications

Software tools ABySS, Velvet, SOAPdenovo, CISA, PGAP
Application De novo sequencing analysis
Diseases Malaria