Breaking the cold chain: solutions for room temperature preservation of mosquitoes leading to high quality reference genomes
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Abstract
Background
The Earth BioGenome Project (EBP) is a global endeavour to produce reference genomes for all described eukaryotic species. The majority of described species are arthropods, which tend to be small and require taxonomic expertise to identify at species level. Therefore, the ability to collect and preserve specimens in a suitable way for long read and Hi-C data generation using very simple approaches with minimal infrastructure will be important in scaling up reference genome generation. Field collections requiring dry ice or dry shippers can be logistically challenging to arrange, are notoriously expensive, and DNA degrades rapidly if ultra-cold temperature is not maintained, which is devastating given how expensive and time consuming fieldwork can be.
Findings
Using Anopheles mosquitoes as an insect representative, we evaluate how well different preservation liquids protect high molecular weight DNA, RNA, and nuclei for Hi-C at room temperature when mosquitoes are held intact versus slightly squished. We find that squished samples stored in 100% ethanol and Allprotect held at room temperature for 1 week result in excellent preservation of both high molecular weight DNA and nuclei for Hi-C. Other tested buffers, including RNAlater, EDTA at several pHs, and dimethyl sulfoxide EDTA salt solution (DESS), performed satisfactorily for long read data generation and RNA retrieval, but less ideally for Hi-C, which may have bigger negative impacts when aiming to generate data for organisms with larger genomes.
Conclusions
We present multiple viable options for room temperature collection and/or shipment for arthropod samples, with direct suggestions for shipment and storage of mosquitoes and similarly sized specimens. Further exploration across a broader range of species will hopefully enable cheaper and more widely available reference genome generation globally.
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AbstractThe Earth BioGenome Project (EBP) is a global endeavour to produce reference genomes for all described eukaryotic species. The majority of described species are arthropods, which tend to be small and require taxonomic expertise to identify to species level. Therefore, the ability to collect and preserve specimens in a suitable way for long read and Hi-C data generation using very simple approaches with minimal infrastructure is certain to be important in scaling up reference genome generation. Using Anopheles mosquitoes as an insect representative we evaluate how well different preservation liquids protect high molecular weight DNA, RNA, and nuclei for Hi-C when mosquitoes are held intact versus slightly squished. We find that squished samples stored in 100% ethanol and Allprotect held at room temperature for one week result in …
AbstractThe Earth BioGenome Project (EBP) is a global endeavour to produce reference genomes for all described eukaryotic species. The majority of described species are arthropods, which tend to be small and require taxonomic expertise to identify to species level. Therefore, the ability to collect and preserve specimens in a suitable way for long read and Hi-C data generation using very simple approaches with minimal infrastructure is certain to be important in scaling up reference genome generation. Using Anopheles mosquitoes as an insect representative we evaluate how well different preservation liquids protect high molecular weight DNA, RNA, and nuclei for Hi-C when mosquitoes are held intact versus slightly squished. We find that squished samples stored in 100% ethanol and Allprotect held at room temperature for one week result in excellent preservation of both high molecular weight DNA and nuclei for Hi-C. Other tested buffers, including RNAlater, EDTA at several pHs, and DMSO Salt Solution (DESS) performed satisfactorily for long read data generation and RNA retrieval, but less ideally for Hi-C, which may have bigger negative impacts when aiming to generate data for organisms with larger genomes. Field collections requiring dry ice or dry shippers can be logistically challenging to arrange, are notoriously expensive, and DNA degrades rapidly if ultra-cold temperature is not maintained, which is devastating given how expensive and time consuming field work can be. Here we present multiple viable options for room temperature collection and/or shipment for arthropod samples. Further exploration across a broader range of species will hopefully enable cheaper and more widely available reference genome generation globally.
This work has been peer reviewed in GigaScience (see https://doi.org/10.1093/gigascience/giag061), which carries out single-anonymized peer review. These reviews are published under a CC-BY 4.0 license and were as follows:
Reviewer 2：
This study uses Anopheles coluzzii as a model to systematically evaluate preservation methods for generating high-quality genomic and Hi-C data, addressing a question of clear practical relevance. The development of approaches that eliminate the need for cold-chain transport is a particularly important step forward for field-based genomics, where maintaining low temperatures is often logistically difficult. In this context, the work provides useful experimental evidence and practical guidance with broad potential value. That said, several aspects of the manuscript would benefit from further clarification and refinement, as outlined below：
- The study is conducted exclusively on Anopheles coluzzii under controlled laboratory conditions. While the results are promising, the extent to which these preservation strategies can be applied to other arthropods remains unclear. Given the substantial diversity in cuticle structure, body size, and physiological properties across taxa, the authors should more explicitly discuss the limitations of extrapolating their findings. Inclusion of additional taxa would be ideal; alternatively, a more thorough discussion of potential constraints would improve the manuscript.
- The evaluation of Hi-C data quality is primarily based on scaffolding performance, including analyses using both high-quality and fragmented assemblies. However, the Hi-C datasets were generated at very high sequencing depth (>100×), which may obscure differences among preservation treatments by compensating for variations in data quality. The authors should clarify how they distinguish genuine preservation effects from depth-related compensation. Additional analyses based on downsampled Hi-C datasets would provide a more realistic assessment of performance under typical sequencing conditions.
- I am particularly interested in the chromosome anchoring rates observed in the Hi-C scaffolding analyses. It would be valuable for the authors to clarify whether Hi-C data generated from different preservation methods lead to differences in chromosome anchoring efficiency, as this is a key indicator of scaffolding quality.
- The manuscript indicates that sequencing data are not yet publicly available due to their inclusion in a larger ENA project. In line with journal policies, the authors should provide a clear plan for data release, including expected timelines and accession numbers where possible. Furthermore, additional methodological details, particularly regarding genome assembly parameters and Hi-C data processing, would improve reproducibility and transparency.
- In Table 1, the inclusion of ULI scaffolding appears somewhat abrupt, as ULI sequencing is not sufficiently introduced or contextualized prior to its presentation in the table. Although its role becomes clearer in the Results section, readers may find it difficult to fully understand its relevance at this stage. The authors may consider introducing ULI sequencing earlier in the Methods or Results, or providing a brief explanation in the table caption, to improve clarity and ensure a more coherent presentation.
This manuscript presents a systematic evaluation of preservation methods for generating high-quality genomic and Hi-C data using Anopheles coluzzii as a model organism. The study addresses an important practical challenge in genomics, particularly for field-based sample collection where optimal preservation conditions are often difficult to achieve. The experimental design is generally well-structured, and the comparison across multiple preservation treatments provides useful insights for the community. However, several aspects of the study require further clarification and improvement. In particular, concerns remain regarding the generalizability of the findings beyond the focal species, the robustness of the statistical analyses, and the interpretation of Hi-C results under very high sequencing coverage. Additionally, issues related to data availability and methodological transparency should be addressed to ensure reproducibility. Addressing these points would substantially strengthen the manuscript. 1.The study is conducted exclusively on Anopheles coluzzii under controlled laboratory conditions. While the results are promising, the applicability of these preservation strategies to other arthropods remains unclear. Given the diversity in cuticle structure, body size, and physiology across taxa, the authors should clarify the extent to which their findings can be generalized. Inclusion of additional taxa or a more explicit discussion of limitations would strengthen the manuscript. 2.The evaluation of Hi-C data quality is based on scaffolding performance, including analyses using both high-quality and fragmented assemblies. However, the Hi-C datasets were generated at very high coverage (>100×), which may mask differences in preservation efficiency. The authors should clarify how they distinguish true preservation effects from sequencing depth-related compensation. Additional analyses using downsampled Hi-C data would provide a more realistic assessment of performance under typical conditions. 3.I am particularly interested in the chromosome anchoring rate of the genome assemblies in the Hi-C scaffolding analysis. It would be valuable for the authors to clarify whether Hi-C data generated using different preservation methods result in differences in chromosome anchoring efficiency.
The column headers “Self scaffolding” and “ULI scaffolding” in Table 1 are not sufficiently clear, making it difficult for readers to fully understand the intended meaning.
The manuscript indicates that sequencing data are not yet publicly available due to their inclusion in a larger ENA project. In line with journal policies, the authors should provide a clear plan for data release, including expected timelines and accession numbers if available. Furthermore, greater detail in the Methods section—particularly regarding assembly parameters and Hi-C processing—would improve reproducibility.
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AbstractThe Earth BioGenome Project (EBP) is a global endeavour to produce reference genomes for all described eukaryotic species. The majority of described species are arthropods, which tend to be small and require taxonomic expertise to identify to species level. Therefore, the ability to collect and preserve specimens in a suitable way for long read and Hi-C data generation using very simple approaches with minimal infrastructure is certain to be important in scaling up reference genome generation. Using Anopheles mosquitoes as an insect representative we evaluate how well different preservation liquids protect high molecular weight DNA, RNA, and nuclei for Hi-C when mosquitoes are held intact versus slightly squished. We find that squished samples stored in 100% ethanol and Allprotect held at room temperature for one week result in …
AbstractThe Earth BioGenome Project (EBP) is a global endeavour to produce reference genomes for all described eukaryotic species. The majority of described species are arthropods, which tend to be small and require taxonomic expertise to identify to species level. Therefore, the ability to collect and preserve specimens in a suitable way for long read and Hi-C data generation using very simple approaches with minimal infrastructure is certain to be important in scaling up reference genome generation. Using Anopheles mosquitoes as an insect representative we evaluate how well different preservation liquids protect high molecular weight DNA, RNA, and nuclei for Hi-C when mosquitoes are held intact versus slightly squished. We find that squished samples stored in 100% ethanol and Allprotect held at room temperature for one week result in excellent preservation of both high molecular weight DNA and nuclei for Hi-C. Other tested buffers, including RNAlater, EDTA at several pHs, and DMSO Salt Solution (DESS) performed satisfactorily for long read data generation and RNA retrieval, but less ideally for Hi-C, which may have bigger negative impacts when aiming to generate data for organisms with larger genomes. Field collections requiring dry ice or dry shippers can be logistically challenging to arrange, are notoriously expensive, and DNA degrades rapidly if ultra-cold temperature is not maintained, which is devastating given how expensive and time consuming field work can be. Here we present multiple viable options for room temperature collection and/or shipment for arthropod samples. Further exploration across a broader range of species will hopefully enable cheaper and more widely available reference genome generation globally.
This work has been peer reviewed in GigaScience (see https://doi.org/10.1093/gigascience/giag061), which carries out single-anonymized peer review. These reviews are published under a CC-BY 4.0 license and were as follows:
Reviewer 1：
This is a straightforward and valuable study. Demonstrating that breaking the cold chain is feasible has strong potential to transform how genomes can be obtained across much of biodiversity. In my view, the broader significance of this advance is somewhat underemphasized in the Introduction (Really just a short mention around lines 72-75). Based on firsthand experience in large field campaigns designed specifically to generate genomes from very small insects, I can attest that much of arthropod biodiversity (especially within small-bodied, hyperdiverse "dark taxa") stands to benefit substantially from approaches like this. The practical importance of scalable, field-friendly preservation solutions cannot be overstated.
Overall, the study is well designed, clearly presented, and addresses a real logistical bottleneck in biodiversity genomics. The conclusions are generally well supported by the data presented. I have a few suggestions that I believe would further strengthen the manuscript and increase its practical value and reproducibility.
Major / Substantive Comments
Framing and impact As above, I recommend strengthening the Introduction's framing of downstream biodiversity applications, especially for small-bodied and taxonomically challenging groups where cold-chain logistics are often the primary limiting factor. The method has particularly high relevance for large-scale efforts targeting hyperdiverse insect groups ("dark taxa"), and this applied significance could be more explicitly highlighted to broaden the paper's audience and appeal.
Practical protocol clarity and reproducibility Because this method is likely to be adopted by field teams, including non-specialists, it would be very helpful to include a concise protocol-style summary or workflow outlining the recommended handling steps, specimen size considerations, and timing constraints. A short practical guide or decision framework would improve reproducibility and uptake.
Relatedly, laying out any known tolerance ranges in this fashion would strengthen the paper, for example, how sensitive outcomes are to delays in processing, temperature variation, or specimen size differences under field conditions.
Preservation benchmarking I suggest including a summary comparison (table or figure) of yield/quality metrics across preservation treatments relative to standard cold-chain approaches. This would make performance differences easier for readers to interpret and apply.
Voucher integrity and downstream taxonomic usability Given that many target organisms will come from taxonomically difficult groups, a short discussion of voucher integrity after treatment would be valuable. Guidance on expected morphological preservation and suitability for downstream taxonomic work would significantly increase the method's usefulness for specimen-based biodiversity genomics workflows.
Methodological Clarification
The instruction to "lightly squish" specimens to compromise the cuticle is practical, but could benefit from additional detail. Does the location of compression affect outcomes? For example, is thoracic compression preferable (to access muscle tissue), or is abdominal disruption sufficient? This may seem like a fine detail, but it matters in practice, particularly because damage to thoracic characters or terminalia can reduce taxonomic value. If there is an optimal or recommended compression location, it would be useful to specify it.
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