June 1, 2021  |  

Long-read assembly of the Aedes aegypti Aag2 cell line genome resolves ancient endogenous viral elements

Transmission of arboviruses such as Dengue Virus by Aedes aegypti causes debilitating disease across the globe. Disease in humans can include severe acute symptoms such as hemorrhagic fever and organ failure, but mosquitoes tolerate high titers of virus in a persistent infection. The mechanisms responsible for this viral tolerance are unclear. Recent publications highlighted the integration of genetic material from non-retroviral RNA viruses into the genome of the host during infection that relies upon endogenous retro-transcriptase activity from transposons. These endogenous viral elements (EVEs) found in the genome are predicted to be ancient, and at least some EVEs are under purifying selection, suggesting they are beneficial to the host. To characterize EVE biogenesis in a tractable system, we sequenced the Ae. aegypti cell line, Aag2, to 58-fold coverage and present a de novo assembly of the genome. The assembly contains 1.7 Gb of genomic and 255 Mb of alternative haplotype specific sequence, consisting of contigs with a N50 of 1.4 Mb; a value that, when compared with other assemblies of the Aedes genus, is from 1-3 orders of magnitude longer. The Aag2 genome is highly repetitive (70%), most of which is classified as transposable elements (60%). We identify EVEs in the genome homologous to a range of extant viruses, many of which cluster in these regions of repetitive DNA. The contiguous assembly allows for more comprehensive identification of the transposable elements and EVEs that are most likely to be lost in assemblies lacking the read length of SMRT Sequencing.


June 1, 2021  |  

Long-read assembly of the Aedes aegypti Aag2 cell line genome resolves ancient endogenous viral elements

Transmission of arboviruses such as Dengue and Zika viruses by Aedes aegypti causes widespread and debilitating disease across the globe. Disease in humans can include severe acute symptoms such as hemorrhagic fever, organ failure, and encephalitis; and yet, mosquitoes tolerate high titers of virus in a persistent infection. The mechanisms responsible for tolerance to viral infection in mosquitoes are still unclear. Recent publications have highlighted the integration of genetic material from non-retroviral RNA viruses into the genome of the host during infection that relies upon endogenous retro-transcriptase activity from transposons. These endogenous viral elements (EVEs) found in the genome are predicted to be ancient and at least some EVEs are under purifying selection, which suggests that they are beneficial to the host. In order characterize EVE biogenesis in a tractable system we sequenced the Ae. aegypti cell line, Aag2, to 58X coverage and here present a de novo assembly of the genome. The assembly consists of 1.7 Gb of genomic and 255 Mb of alternative haplotype specific sequence, made up of contigs with a N50 of 1.4 Mb; a value that, when compared with other assemblies of the Aedes genus, is from 1-3 orders of magnitude longer. The Aag2 genome is highly repetitive (70%), most of which is classified as transposable elements (60%). We identify a plethora of EVEs in the genome homologous to a diverse range of extant viruses, many of which cluster in these regions of highly repetitive DNA. The highly contiguous nature of this assembly allows for a more comprehensive identification of the transposable elements and EVEs that are most likely to be lost in assemblies lacking the read length of SMRT Sequencing. Transmission of arboviruses such as Dengue Virus by Aedes aegypti causes widespread and debilitating disease across the globe. Disease in humans can include severe acute symptoms such as hemorrhagic fever, organ failure, and encephalitis; and yet, mosquitoes tolerate high titers of virus in a persistent infection. The mechanisms responsible for tolerance to viral infection in mosquitoes are still unclear. Recent publications have highlighted the integration of genetic material from non-retroviral RNA viruses into the genome of the host during infection that relies upon endogenous retro-transcriptase activity from transposons. These endogenous viral elements (EVEs) found in the genome are predicted to be ancient and at least some EVEs are under purifying selection, which suggests that they are beneficial to the host. In order characterize EVE biogenesis in a tractable system we sequenced the Ae. aegypti cell line, Aag2, to 58X coverage and here present a de novo assembly of the genome. The assembly consists of 1.7 Gb of genomic and 255 Mb of alternative haplotype specific sequence, made up of contigs with a N50 of 1.4 Mb; a value that, when compared with other assemblies of the Aedes genus, is from 1-3 orders of magnitude longer. The Aag2 genome is highly repetitive (70%), most of which is classified as transposable elements (60%). We identify a plethora of EVEs in the genome homologous to a diverse range of extant viruses, many of which cluster in these regions of highly repetitive DNA. The highly contiguous nature of this assembly allows for a more comprehensive identification of the transposable elements and EVEs that are most likely to be lost in assemblies lacking the read length of SMRT Sequencing. Transmission of arboviruses such as Dengue Virus by Aedes aegypti causes widespread and debilitating disease across the globe. Disease in humans can include severe acute symptoms such as hemorrhagic fever, organ failure, and encephalitis; and yet, mosquitoes tolerate high titers of virus in a persistent infection. The mechanisms responsible for tolerance to viral infection in mosquitoes are still unclear.


June 1, 2021  |  

A high-quality genome assembly of SMRT Sequences reveals long-range haplotype structure in the diploid mosquito Aedes aegypti

Aedes aegypti is a tropical and subtropical mosquito vector for Zika, yellow fever, dengue fever, chikungunya, and other diseases. The outbreak of Zika in the Americas, which can cause microcephaly in the fetus of infected women, adds urgency to the need for a high-quality reference genome in order to better understand the organism’s biology and its role in transmitting human disease. We describe the first diploid assembly of an insect genome, using SMRT sequencing and the open-source assembler FALCON-Unzip. This assembly has high contiguity (contig N50 1.3 Mb), is more complete than previous assemblies (Length 1.45 Gb with 87% BUSCO genes complete), and is high quality (mean base >QV30). Long-range haplotype structure, in some cases encompassing more than 4 Mb of extremely divergent homologous sequence, is resolved using a combination of the FALCON-Unzip assembler, genome annotation, coverage depth, and pairwise nucleotide alignments.


June 1, 2021  |  

A high-quality genome assembly of SMRT sequences reveals long range haplotype structure in the diploid mosquito Aedes aegypti

Aedes aegypti is a tropical and subtropical mosquito vector for Zika, yellow fever, dengue fever, and chikungunya. We describe the first diploid assembly of an insect genome, using SMRT Sequencing and the open-source assembler FALCON-Unzip. This assembly has high contiguity (contig N50 1.3 Mb), is more complete than previous assemblies (Length 1.45 Gb with 87% BUSCO genes complete), and is high quality (mean base >QV30 after polishing). Long-range haplotype structure, in some cases encompassing more than 4 Mb of extremely divergent homologous sequence with dramatic differences in coding sequence content, is resolved using a combination of the FALCON-Unzip assembler, genome annotation, coverage depth, and pairwise nucleotide alignments.


April 21, 2020  |  

Insect genomes: progress and challenges.

In the wake of constant improvements in sequencing technologies, numerous insect genomes have been sequenced. Currently, 1219 insect genome-sequencing projects have been registered with the National Center for Biotechnology Information, including 401 that have genome assemblies and 155 with an official gene set of annotated protein-coding genes. Comparative genomics analysis showed that the expansion or contraction of gene families was associated with well-studied physiological traits such as immune system, metabolic detoxification, parasitism and polyphagy in insects. Here, we summarize the progress of insect genome sequencing, with an emphasis on how this impacts research on pest control. We begin with a brief introduction to the basic concepts of genome assembly, annotation and metrics for evaluating the quality of draft assemblies. We then provide an overview of genome information for numerous insect species, highlighting examples from prominent model organisms, agricultural pests and disease vectors. We also introduce the major insect genome databases. The increasing availability of insect genomic resources is beneficial for developing alternative pest control methods. However, many opportunities remain for developing data-mining tools that make maximal use of the available insect genome resources. Although rapid progress has been achieved, many challenges remain in the field of insect genomics. © 2019 The Royal Entomological Society.


April 21, 2020  |  

Evolutionary superscaffolding and chromosome anchoring to improve Anopheles genome assemblies

Background New sequencing technologies have lowered financial barriers to whole genome sequencing, but resulting assemblies are often fragmented and far from textquoteleftfinishedtextquoteright. Updating multi-scaffold drafts to chromosome-level status can be achieved through experimental mapping or re-sequencing efforts. Avoiding the costs associated with such approaches, comparative genomic analysis of gene order conservation (synteny) to predict scaffold neighbours (adjacencies) offers a potentially useful complementary method for improving draft assemblies.Results We employed three gene synteny-based methods applied to 21 Anopheles mosquito assemblies to produce consensus sets of scaffold adjacencies. For subsets of the assemblies we integrated these with additional supporting data to confirm and complement the synteny-based adjacencies: six with physical mapping data that anchor scaffolds to chromosome locations, 13 with paired-end RNA sequencing (RNAseq) data, and three with new assemblies based on re-scaffolding or Pacific Biosciences long-read data. Our combined analyses produced 20 new superscaffolded assemblies with improved contiguities: seven for which assignments of non-anchored scaffolds to chromosome arms span more than 75% of the assemblies, and a further seven with chromosome anchoring including an 88% anchored Anopheles arabiensis assembly and, respectively, 73% and 84% anchored assemblies with comprehensively updated cytogenetic photomaps for Anopheles funestus and Anopheles stephensi.Conclusions Experimental data from probe mapping, RNAseq, or long-read technologies, where available, all contribute to successful upgrading of draft assemblies. Our comparisons show that gene synteny-based computational methods represent a valuable alternative or complementary approach. Our improved Anopheles reference assemblies highlight the utility of applying comparative genomics approaches to improve community genomic resources.ADADSEQAGOAGOUTI-basedAGOUTIannotated genome optimization using transcriptome information toolALNalignment-basedCAMSAcomparative analysis and merging of scaffold assemblies toolDPdynamic programmingFISHfluorescence in situ hybridizationGAGOS-ASMGOS-ASMGene order scaffold assemblerKbpkilobasepairsMbpmegabasepairsOSORTHOSTITCHPacBioPacific BiosciencesPBPacBio-basedPHYphysical-mapping-basedRNAseqRNA sequencingQTLquantitative trait lociSYNsynteny-based.


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