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Updated: Jul 20, 2025
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Ultra-long Read Sequencing for Whole Genomic DNA Analysis
Published on: March 15, 2019
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Exploration of whole genome amplification generated chimeric sequences in long-read sequencing data
Na Lu1, Yi Qiao1, Pengfei An1,2
1State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China.
Briefings in Bioinformatics
|August 2, 2023
Summary
Chimeric sequences formed during whole genome amplification interfere with long-read sequencing. A new pipeline, 3rd-ChimeraMiner, identifies and corrects these artifacts, improving structural variation analysis in amplified DNA.
Area of Science:
- Genomics
- Molecular Biology
- Bioinformatics
Background:
- Multiple displacement amplification (MDA) is a common whole genome amplification technique.
- MDA generates high molecular weight DNA suitable for long-read sequencing (>20 kb).
- Chimeric sequences (chimeras) are structural errors in MDA data that hinder analysis, but their impact on long-read sequencing is unclear.
Purpose of the Study:
- To investigate the formation and impact of chimeras in MDA-amplified DNA sequenced using long-read technology.
- To develop a computational tool for identifying and correcting chimeras in long-read sequencing data.
- To improve the accuracy of structural variation analysis in MDA-generated samples.
Main Methods:
- Sequencing of phi29 DNA polymerase-mediated MDA amplicons on the PacBio platform.
- Development and application of the 3rd-ChimeraMiner pipeline for chimera recognition and restoration.
- Analysis of multiple long-read and high-fidelity long-read datasets with varying amplification folds.
Main Results:
- Mis-priming events, leading to chimeras, occur frequently during MDA, increasing from 42% to over 78% with amplification.
- 99.92% of identified chimeras are artifacts of the MDA process, not present in the original genome.
- Restoring chimeras reduced false-positive structural variants, including 97% of inversions on average.
Conclusions:
- Chimeras are a significant artifact in MDA-amplified DNA, particularly impacting long-read sequencing data analysis.
- The 3rd-ChimeraMiner pipeline effectively quantifies and corrects chimera-induced errors.
- This tool enhances the reliability of structural variation analysis for MDA-amplified samples.
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