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Amplicon Sequencing using the Long-Read Sequencing Technologies
Published on: August 29, 2025
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Pitfalls of haplotype phasing from amplicon-based long-read sequencing.
Thomas W Laver1, Richard C Caswell1, Karen A Moore2
1University of Exeter Medical School, RILD Building, Barrack Road, Exeter EX2 5DW, UK.
Scientific Reports
|February 18, 2016
Summary
Long-read sequencing technologies from PacBio and Oxford Nanopore can phase distant variants. However, PCR chimeras and alignment bias are critical pitfalls in amplicon-based phasing that must be addressed.
Area of Science:
- Genomics
- Molecular Biology
- Bioinformatics
Background:
- Long-read sequencing technologies, including Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT), enable phasing of variants over kilobase distances.
- Accurate phasing of genetic variants is crucial for understanding disease associations and complex genetic traits.
Purpose of the Study:
- To evaluate the utility of long-read sequencing for phasing distant variants using amplicon-based approaches.
- To identify and characterize potential methodological pitfalls in variant phasing with PacBio and ONT technologies.
Main Methods:
- Long-range PCR amplification of target regions in the RET gene.
- Sequencing of amplicons using both PacBio and ONT platforms.
- Re-analysis of publicly available ONT sequencing data for CYP2D6 and HLA loci phasing.
- Bioinformatic analysis to assess variant phasing accuracy and identify artifacts.
Main Results:
- Successful phasing of two variants 9 kb apart in the RET gene was demonstrated.
- Identification of PCR-chimera formation as a significant artifact during amplicon amplification.
- Detection of reference alignment bias impacting variant phasing accuracy.
- Re-analysis revealed potential issues in previously reported phasing of CYP2D6 and HLA haplotypes.
Conclusions:
- Amplicon-based long-read sequencing offers potential for phasing distant variants but is susceptible to methodological pitfalls.
- PCR-chimera formation and reference alignment bias can lead to inaccurate phasing results.
- Careful experimental design and bioinformatic analysis are essential to overcome these limitations and fully leverage long-read sequencing for variant phasing.
