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As genomic research advances rapidly and accurately analyzing complex genomes is a key challenge. Bacterial artificial chromosomes (BACs) stably carry large DNA fragments, making them essential for physical mapping, gene localization, analysis of complex gene families, and genome assembly.
Why Is BAC End Sequencing Valuable?
BAC end sequencing (BES) determines the sequences at both ends of BAC inserts, providing critical anchoring information. This enables integration of large DNA fragments with reference genomes, facilitates assembly, fills gaps, and detects structural variations. In complex or highly repetitive genomes, long-span paired-end sequences serve as essential positional references for whole-genome studies.
How Does BAC End Sequencing Work?
The principle of BES is to use end sequences of BAC inserts as anchors. With insert sizes ranging from 100 to 300 kb, these sequences span large repetitive regions, facilitating the localization of complex genomic areas, verification of assembly accuracy, and identification of structural variations.
What Are Its Applications?
BES assists genome assembly by detecting and correcting errors, provides markers for physical map construction, supports gene localization in crops and model organisms, and detects exogenous contamination from chloroplast, mitochondrial, or bacterial DNA to improve genomic accuracy. Compared to traditional Sanger sequencing, modern BES combines high-throughput and long-read sequencing platforms, enabling faster, more informative, and cost-effective data acquisition.
Creative Biogene has established a comprehensive BAC end sequencing platform that covers the entire workflow from sample preparation to data analysis. Aware of the challenges in BAC sequencing, we have systematically optimized DNA extraction, sequencing reactions, and data processing to deliver high-quality, reproducible, and application-ready results.
Clone Matrix Pooling Strategy
BAC clones are arranged in a two-dimensional matrix, with clones from each row and column pooled separately to form sub-pools. Each sub-pool is sequenced to generate pooled sequence data. By cross-referencing row and column pool sequences, sequences can be accurately assigned to intersecting clones, resolving the end sequences of each BAC clone. This strategy significantly increases throughput while avoiding the high cost and low efficiency of individual clone sequencing.
Long-Read Sequencing Technology
Leveraging third-generation sequencing platforms, the long-read advantage is fully utilized to obtain average BAC end sequences of 2–3 kb. This enables the coverage of complex, repetitive regions and high-GC areas, thereby overcoming the limitations of short-read sequencing. High-accuracy sequencing ensures the reliable mapping of paired ends, providing precise data for genome assembly and analysis of structural variations.
Paired-End Mapping and Analysis
Using both end sequences and insert length information, BAC clones are precisely mapped to the reference genome. This allows the identification of assembly errors, inversions, translocations, and other structural variations, providing comprehensive BAC mapping information for gene localization, functional studies, and library integration.
Data Processing and Quality Control
Raw sequencing data are processed to remove noise and select high-quality bases, eliminating interference from residual dyes and secondary structures. Automated analysis pipelines, combined with manual curation, ensure the high accuracy of paired-end assignments and mapping. Detailed reports are provided, including end sequences, insert lengths, mapping results, and data quality evaluations.
1High-quality DNA extraction: Ensures sufficient purity and concentration from low-copy BAC clones to meet downstream sequencing requirements.
2Multi-platform support: Integrates high-throughput Illumina sequencing with long-read platforms, balancing coverage and read-length advantages.
3Extended end lengths: End lengths exceeding 1–2 kb provide richer information compared to conventional methods.
4Standardized data analysis: Includes quality control, contamination checks, and reference genome alignment for reliable results.
5Broad applications: Suitable for genome assembly refinement, studies of complex species, BAC library integration, and gene mapping.
BAC end sequencing, with its unique long-span anchoring advantage, plays a crucial and irreplaceable role in genomic research. Leveraging advanced sequencing platforms and optimized experimental workflows, Creative Biogene provides high-quality BAC end sequencing services to address critical challenges in genome assembly, structural variation detection, and BAC clone mapping. We aim to be a trusted partner for research and industry clients worldwide, accelerating progress in genome research and its applications.
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Q1: What are the advantages of BAC end sequencing compared to whole-genome sequencing?
A1: Whole-genome sequencing provides overall coverage, whereas BAC end sequencing delivers specific long-fragment anchoring information. Paired-end sequences can span repetitive regions and enable precise localization, making BES particularly valuable for genome assembly and structural variation studies.
Q2: What is the typical read length of end sequences?
A2: Traditional NGS platforms generally provide reads shorter than 1 kb. Using our long-read strategy, end sequences of 2–3 kb can be reliably obtained, significantly enhancing resolution.
Q3: What genomic issues can BAC end sequencing address?
A3: BAC end sequencing is primarily used to verify genome assemblies, map BAC clones, detect exogenous contamination, and evaluate structural variations. It is especially important for complex or highly repetitive genomes.
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