A Workflow to Improve Variant Calling Accuracy in Molecular Barcoded Sequencing Reads

Area of Science:

• Genomics
• Molecular Biology
• Bioinformatics

Background:

• Multiplexed molecular barcoded amplicon sequencing enhances low-frequency variant detection sensitivity.
• Molecular barcodes correct amplification biases and sequencing errors.
• High false-positive rates in repetitive regions challenge variant calling accuracy.

Purpose of the Study:

• To develop and validate a generic workflow for improving variant calling accuracy using molecular barcoded sequencing data.
• To specifically address false positives in homopolymer and repetitive regions.
• To assess the efficacy of a base score correction method on targeted sequencing data.

Main Methods:

• A generic workflow applying a base score correction method to duplicate or overlapping read pairs from molecular barcoded sequencing.
• Analysis focused on three specific regions within a custom QIAseq targeted DNA panel.
• Comparison of uncorrected and corrected datasets against NIST reference calls and Macrogen exome data.

Main Results:

• The base score correction workflow effectively reduced the false-positive rate in variant calls.
• All identified false positives were removed by the correction method.
• True positive calls were retained in the dataset after correction.
• The workflow demonstrated improved accuracy in repetitive genomic regions.

Conclusions:

• The proposed workflow incorporating base correction of molecular barcoded sequencing data significantly improves variant calling accuracy.
• This method is particularly effective in reducing false positives in challenging genomic regions with repetitive sequence motifs.
• The workflow is applicable to germline sequencing for enhanced genomic analysis.