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Updated: Jul 16, 2025

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

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Evaluating restriction enzyme selection for reduced representation sequencing in conservation genomics.

Ainhoa López^1,2, Carlos Carreras^1,2, Marta Pascual^1,2

¹Departament de Genètica, Microbiologia i Estadística, Facultat de Biologia, Universitat de Barcelona (UB), Barcelona, Spain.

Molecular Ecology Resources

|September 14, 2023

Summary

Restriction enzyme choice impacts genome representation in conservation genomics. Genotyping-by-Sequencing (GBS) analysis revealed enzyme-specific biases, emphasizing the need for careful selection and high-quality genomes for accurate population studies.

Keywords:

GBS SNPs conservation genomics functional annotation reduced representation sequencing reference genome restriction enzymes

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High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

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Published on: October 5, 2018

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Novel Sequence Discovery by Subtractive Genomics

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Published on: January 25, 2019

Area of Science:

Genomics
Conservation Biology
Bioinformatics

Background:

Reduced representation sequencing, like Genotyping-by-Sequencing (GBS), is crucial for conservation genomics in non-model organisms.
The distribution of sequenced loci may be influenced by the restriction enzyme's recognition site GC content, potentially biasing genome representation.

Purpose of the Study:

To evaluate the distribution and functional composition of loci generated by GBS.
To assess the impact of different restriction enzymes (EcoT22I and ApeKI) on genome representation in fish and sea urchin species.
To investigate potential biases in sampling exonic, intronic, and intergenic regions.

Main Methods:

Applied GBS to two fish species (Symphodus ocellatus, S. tinca) and two sea urchin species (Paracentrotus lividus, Arbacia lixula).
Mapped sequenced loci to the closest available reference genomes (Labrus bergylta, Strongylocentrotus purpuratus).
Classified loci and analyzed functions using Gene Ontology (GO) terms; simulated enzyme effects on reference genomes.

Main Results:

Detected enzyme-dependent enrichment of loci in exonic or intergenic regions in both simulated and experimental data.
Found no significant differences between total and candidate loci for adaptation in empirical data.
Identified shared, general functions across all four species for the mapped loci.

Conclusions:

Restriction enzyme choice significantly influences genome representation in GBS studies.
High-quality, annotated reference genomes are essential for accurate conservation genomic analyses.
Enzyme selection biases need consideration to avoid misinterpreting population structure and local adaptation signals.