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BAMClipper: removing primers from alignments to minimize false-negative mutations in amplicon next-generation
Chun Hang Au1, Dona N Ho1, Ava Kwong2,3,4
1Division of Molecular Pathology, Department of Pathology, Hong Kong Sanatorium & Hospital, Happy Valley, Hong Kong SAR, China.
Scientific Reports
|May 10, 2017
Summary
BAMClipper improves genetic variation detection in next-generation sequencing (NGS) by removing primer sequences post-mapping. This method accurately identifies mutations missed by conventional primer trimming, enhancing variant allele frequency accuracy.
Area of Science:
- Genomics
- Bioinformatics
- Molecular Biology
Background:
- Amplicon-based next-generation sequencing (NGS) is crucial for genetic variation detection.
- Current methods often trim primer sequences before read mapping, potentially affecting accuracy.
- Primer handling strategies can impact mutation detection sensitivity and variant allele frequency estimation.
Purpose of the Study:
- Introduce BAMClipper, a novel tool for primer sequence removal after read mapping in NGS data.
- Evaluate the impact of primer handling approaches on mutation detection accuracy.
- Compare BAMClipper's performance against conventional primer trimming methods.
Main Methods:
- Developed BAMClipper to soft-clip primer sequences in SAM/BAM alignments post-mapping.
- Analyzed real NGS datasets from human samples with known BRCA1/BRCA2 mutations.
- Utilized over 130,000 simulated NGS datasets with unique mutations for validation.
Main Results:
- BAMClipper detected a BRCA1 deletion (c.1620_1636del) missed by conventional methods due to edge effects.
- Conventional primer trimming led to high false-negative rates and significant under-estimation of variant allele frequencies (17-82%).
- BAMClipper successfully detected all 7 known mutations across real and simulated datasets, outperforming standard approaches.
Conclusions:
- BAMClipper enhances the accuracy of genetic variation detection in amplicon-based NGS.
- The post-mapping primer removal strategy overcomes limitations of pre-mapping trimming, improving sensitivity and VAF accuracy.
- BAMClipper offers a robust and efficient solution for primer handling in NGS data analysis.
