De novosequencing, diploid assembly, and annotation of the black carpenter ant,Camponotus pennsylvanicus, and its symbionts by one person for $1000, using nanopore sequencing

Abstract

The black carpenter ant (Camponotus pennsylvanicus) is a pest species found widely throughout North America east of the Rocky Mountains. Its frequent infestation in human buildings justifies close genetic examination and its large size and small genome make it ideal for individual sequencing. From a single individual I used long-read nanopore sequencing to assemble a genome of 306 Mb, with an N50 of 565 Kb, and 60X coverage, with quality assessed by a 97.0% BUSCO score, improving upon related ant assemblies. The reads provided secondary information in the form of parasitic and symbiont genomes, as well as epigenetic information. I assembled a complete Wolbachia bacterial isolate with a genome size of 1.2 Mb and 76X coverage, as well as a commensal bacterium of the carpenter ant tribe, the species-specific symbiont Blochmannia pennsylvanicus, at 791 Kb, 2400X coverage, which matched to within 200 bp of its previously published reference. I also produced a complete mitochondrial genome with over 5000X coverage, revealing minor rearrangements and the first assembly for this species. DNA methylation and hydroxymethylation was measured simultaneously at whole genome, base-pair resolution level from the same nanopore reads and confirmed extremely low levels seen in the Formicidae family of Hymenoptera. A phased diploid assembly was built, revealing a moderate level of heterozygosity, with 0.16% of bases having biallelic SNPs from the two parental haplotypes. Protein prediction yielded 20,209 putative amino acid sequences and annotation identified 86% matched to previously known proteins. All assemblies were derived from a single Minion flow cell generating 20 Gb of sequence for a cost of $1047 including all consumable reagents. Adding fixed costs for required equipment and batch costs for reagents brings the cost to assemble an ant-sized genome to less than $5000. Complete analysis was performed in under 1 week on a commodity computer desktop with 64 Gb memory.

Impact of host demography and evolutionary history on endosymbiont molecular evolution: A test in carpenter ants (genusCamponotus) and theirBlochmanniaendosymbionts

“…We decided to assemble the genome of a member of the subgenus Camponotus because our research group is focusing on this subgenus in this and several other projects. In April 2022, a somewhat contiguous genome of C. pennsylvanicus (a member of the subgenus Camponotus ) was reported in a preprint (Faulk, 2022 ); our genome is assembled into scaffolds ~10× more contiguous than that of this reported C. pennsylvanicus genome (Table 3 ).…”

“…was reported in a preprint (Faulk, 2022); our genome is assembled into scaffolds ~10× more contiguous than that of this reported C. pennsylvanicus genome (Table 3).…”

Sequencing Bait: Nuclear and Mitogenome Assembly of an Abundant Coastal Tropical and Subtropical Fish,Atherinomorus stipes

“…One potential drawback of lcWGS is the need for a reference genome ( Therkildsen and Palumbi 2017 ). Yet, with improved accuracy in third-generation sequencing technology, including Pacific Biosciences (PacBio) and Oxford Nanopore (ONT) sequencing, it is feasible and affordable to prepare a de novo genome or transcriptome for nonmodel organisms ( Gatter et al 2020 ; Faulk 2022 ). Incorporating short read data can additionally mitigate long read sequencing error rates [now approximately 13% for PacBio ( Ardui et al 2018 ) less than 5% for ONT ( Dohm et al 2020 )] and improve final assembly quality ( Michael et al 2018 ; Shin et al 2019 ; Warren et al 2019 ; Gatter et al 2020 ; Chen et al 2021 ).…”