FalseAmpHunter: A Bioinformatics Pipeline for Detecting and Characterizing False Amplicons in PCR
Muhammad Shoaib Akhtar, Mian Sahib Zar, Abdul Rehman Haris, Samia TahirIntroduction:
Polymerase chain reaction (PCR) is a widely used molecular biology technique; however, it remains highly susceptible to non-specific primer binding, leading to off-target amplification, particularly in genomic regions with extensive sequence similarity. Such off-target amplification can generate false amplicons that are difficult to detect using conventional quality control methods and may lead to erroneous downstream interpretations. Here, we present FalseAmpHunter, a pipeline designed to detect, assemble, and characterize false amplicons from paired-end next-generation sequencing (NGS) data generated by PCR amplification.
Methods:
FalseAmpHunter reconstructs candidate amplicons, maps them genome-wide, evaluates primer-binding orientation, and distinguishes true target amplification from paralog-driven off-target products and sequencing artifacts. We validated FalseAmpHunter using a synthetic dataset derived from in silico PCR of paralogous olfactory receptor (OR) genes.
Results:
The pipeline successfully identified both the intended target and false amplicons originating from paralogous loci, while excluding a random decoy control. FalseAmpHunter provides a systematic and transparent solution for investigating false PCR amplification events and is applicable to assay development, primer validation, and troubleshooting of targeted sequencing experiments.
Discussion:
By transforming raw sequencing data into interpretable genomic evidence, FalseAmpHunter enhances confidence in false amplicon discovery in PCR-based analyses, particularly in paralog- rich genomic contexts.
Conclusion:
FalseAmpHunter provides an easy and reproducible workflow for identifying false amplicons in off-target amplification products. The pipeline is accessible online at: https://github.com/xoaib4/FalseAmpHunter.