Service Overview

CD Genomics integrates cutting-edge third-generation sequencing technology (PacBio HiFi) with proprietary micro-library preparation techniques to offer high-precision de novo sequencing solutions tailored specifically for the study of minute insect genomes. This service adeptly addresses common challenges associated with diminutive insect specimens (diameter 2-5mm), such as difficulty in DNA extraction and high heterozygosity. By requiring a minimal input of only 20ng of DNA, we successfully achieve the construction of HiFi libraries and precise genome assembly. The results are high-quality genomic data, yielding a Contig N50 of up to 1Mb and a BUSCO completeness exceeding 95%. This innovative solution is widely applicable across disciplines, including agricultural pest management, biodiversity conservation, and the study of insect evolutionary mechanisms.

Challenges in De Novo Sequencing of Insect Genomes

1. Small Insect Specimens: Insects such as parasitic wasps and fruit flies yield insufficient amounts of DNA/RNA due to their diminutive size, posing challenges for library construction. Traditional methods often require pooling multiple specimens, which can lead to issues such as chimeric assembly and genome redundancy.

2. Impact of GC/AT Content: Insect genomes typically exhibit low GC content and high AT content, which can affect the stability and consistency of sequencing data, thereby increasing the difficulty of genome assembly.

3. High Heterozygosity in Assemblies: Insect populations are characterized by high genetic diversity, often resulting in assemblies with numerous heterozygous sequences. This necessitates complex processes for redundancy removal and sequence alignment.

4. Lack of Reference Genome Databases: Although some reference genomes for insects have been published, they are insufficient when considering the vast diversity of insect species. This paucity presents challenges for genome annotation and analysis.

To address these challenges and enhance the quality and efficiency of genome assembly, researchers have undertaken the following initiatives:

1. Improving efficient DNA extraction methods;

2. Refining micro-library preparation techniques;

3. Optimizing Hi-C experimental workflows and assembly strategies;

4. Developing more robust bioinformatics tools to tackle high heterozygosity and genomic complexity.

Technical Advantages of Micro-Insects De Novo Genome Sequencing

1. Micro-Library Preparation Technology

• Ultra-Low DNA Requirement: This approach supports single-specimen insect samples, requiring only 20ng of DNA to construct HiFi libraries, overcoming the traditional limitation of needing 5μg of DNA.
• Stable Data Output: Yields from single cells exceed 94Gb, with sequencing data meeting a standardization rate of over 100%, reaching up to 121.21%, ensuring stable sequencing of highly heterozygous samples.
• Avoidance of Chimeric Assemblies: The single-specimen sequencing technique eliminates genome redundancy and errors caused by pooled samples. Additionally, Hi-C assisted assemblies have a scaffold mounting rate of over 95%, achieving chromosomal-level precision.

2. Complex Genome Processing Capability

• Optimization for High Repetition/High Heterozygosity: By combining long-read sequencing with iterative assembly algorithms, this technology effectively resolves complex regions such as telomeres and centromeres, supporting telomere-to-telomere (T2T) genome assemblies.
• Haplotype Analysis: Enables differentiation between paternal and maternal genetic information, revealing hybrid vigor and functional differences in alleles.

3. Comprehensive Technical Support

• From Sample to Annotation: The service provides an end-to-end solution, encompassing DNA extraction, library construction, third-generation sequencing, Hi-C assisted assembly, functional annotation, and evolutionary analysis.
• Customized Solutions: Tailor-made library strategies are developed for specific species, such as parasitic wasps, fruit flies, and thrips, supporting pan-genomic and exceptionally large genomic projects (≥10Gb).

Empirical Data

1. Actual Project Outcomes

Analysis of empirical data suggests a correlation between DNA input quantity and library performance. Optimal library yields are achievable with DNA inputs around 100ng, with an average yield ratio exceeding 100%.

2. Actual Assembly Results

From the genome assembly results, it is evident that the sequencing data obtained via micro-library preparation sufficiently meets the requirements for de novo insect genome construction. The assembled genome Contig N50 reaches up to 1Mb, and BUSCO assessments consistently exceed 95%, indicating satisfactory genome integrity.

3. Actual Scaffold Mounting Results

The Hi-C scaffold mounting results, derived from micro-library preparations, are clear and effectively circumvent the chimeric phenomena typically caused by mixed-sample sequencing. Chromosomal-level assembly achieves a scaffold mounting rate of over 95%.

Applications of Micro-Insects De Novo Genome Sequencing

Our services are particularly designed for small insect specimens with diameters ranging from 2 to 5mm, such as ants, thrips, and brown planthoppers. Traditional genome sequencing methods for these insects often struggle with limitations related to sample quantity and assembly quality. CD Genomics' micro-library preparation technology effectively addresses these challenges, providing innovative solutions for small insect genome research.

1. Small Insect Specimens: For insect specimens within the range of 2-5mm in diameter, such as parasitic wasps, fruit flies, and thrips, our technology reduces the DNA requirement dramatically. While traditional third-generation library construction might demand about 5μg of DNA for a data output of 30G, our micro-library preparation requires only about 100ng of DNA, approximately 1/30th of the conventional amount.

2. Difficult-to-Culture and Mixed Sequencing Samples: While traditional methods may involve sequencing pooled samples, some insect specimens are challenging to culture and collect in large quantities, creating an experimental bottleneck. Our micro-library preparation allows for effective sequencing from single-specimen extractions, overcoming this challenge efficiently.

3. High Heterozygosity Samples: In species with inherently complex genomic structures or high heterozygosity, traditional methods that mix multiple samples for library preparation often lead to genome assemblies that exceed expected sizes and cannot be corrected through bioinformatics approaches. Utilizing single-specimen micro-library preparation overcomes these technical limitations.

4. Other Species: Our approach is adaptable for other small organisms, such as aquatic microorganisms, aquatic plants, and plankton, enabling genomic research on a variety of diminutive species.

Micro-Insects De Novo Genome Sequencing Service Workflow

1. Sample Evaluation: Upon receiving the sample, we assess DNA integrity using the Agilent 4200 system, ensuring a DV200 score of over 80% to meet library preparation standards.

2. Micro-Library Preparation and Sequencing: Utilizing the PacBio SMRTbell library preparation kit, we optimize the enzymatic digestion and ligation steps to preferentially capture long fragments of approximately 14kb.

3. Data Assembly and Optimization: Initial assembly and heterozygosity reduction are performed using Wtdbg2 and HaploMerger2. Hi-C data is then integrated to enhance chromosomal continuity and scaffold mounting.

4. Annotation and Delivery: Gene function annotation is conducted using databases such as NCBI, KEGG, and SwissProt. We provide a comprehensive delivery package, including a Contig N50 and BUSCO assessment report, along with visualized genome circos plots.

Technical Workflow for Insect Micro-Library Sequencing

Sample Requirements

Case Study of Micro-Insects De Novo Genome Sequencing

On May 2, 2024, a research team led by Professor Haijun Xu at the Institute of Insect Sciences, published an article in Scientific Data entitled "Chromosome-level Genome Assembly of the Bethylid Ectoparasitoid Wasp Sclerodermus sp. 'alternatusi'." This study delves into the genomic intricacies of the wasp Sclerodermus sp. 'alternatusi', a member of the order Hymenoptera.

Research Objective:

The primary aim of the study was to establish a high-quality chromosome-level genome assembly for Sclerodermus sp. 'alternatusi'. This would facilitate a deeper understanding of its biological characteristics and provide fundamental data for future functional genomics research.

Research Methods:

• Genome Sequencing: The research utilized PacBio HiFi sequencing technology to obtain high-fidelity, long-read data.
• Hi-C Assisted Assembly: Hi-C technology was employed to optimize the assembly results, ensuring high-quality, chromosome-level assembly.
• Gene Annotation: The assembled genome underwent gene prediction and functional annotation to extract in-depth genomic information.

Research Findings:

• Genome Size: The assembled genome was approximately 162.3 Mb, reflecting high assembly quality.
• Gene Prediction: A total of 10,204 protein-coding genes were annotated, providing a valuable resource for functional genomics studies of the wasp.
• Repeat Sequence Analysis: The study identified repeat sequences constituting 14.17% of the genome, offering significant insights into its genomic structure.

Chromosome-level genome assembly of the bethylid ectoparasitoid wasp Sclerodermus sp. 'alternatusi'. (Wan, Y., et al. 2024).

Why Choose CD Genomics

• Expert Team: Our team possesses extensive expertise in insect genome research and offers tailored services to meet your specific research needs.
• Advanced Technology: We employ state-of-the-art micro-library and third-generation HiFi sequencing technologies to ensure the accuracy and reliability of results.
• Customer Support: We provide comprehensive technical support and consultation services to assist you in overcoming any challenges in your research.

Frequently Asked Questions of Micro-Insects De Novo Genome Sequencing

1. Can you handle samples with high host contamination?

• Yes, we offer host DNA removal techniques, suitable for studies involving insect parasites or symbiotic microorganisms.

2. Do you provide functional validation services?

• We offer complementary services such as single-cell transcriptomics and CRISPR editing platforms to validate the functions of key genes.

Reference

1. Wan, Y., Wu, HJ., Yang, JP. et al. Chromosome-level genome assembly of the bethylid ectoparasitoid wasp Sclerodermus sp. 'alternatusi'. Sci Data 11, 438 (2024). https://doi.org/10.1038/s41597-024-03278-0