Abstract
BackgroundMosaic mutations contribute to numerous human disorders. As such, the identification and precise quantification of mosaic mutations is essential for a wide range of research applications, clinical diagnoses, and early detection of cancers. Currently, the low-throughput nature of single allele assays (e.g., allele-specific ddPCR) commonly used for genotyping known mutations at very low alternate allelic fractions (AAFs) have limited the integration of low-level mosaic analyses into clinical and research applications. The growing importance of mosaic mutations requires a more rapid, low-cost solution for mutation detection and validation.MethodsTo overcome these limitations, we developed Multiple Independent Primer PCR Sequencing (MIPP-Seq) which combines the power of ultra-deep sequencing and truly independent assays. The accuracy of MIPP-seq to quantifiable detect and measure extremely low allelic fractions was assessed using a combination of SNVs, insertions, and deletions at known allelic fractions in blood and brain derived DNA samples.ResultsThe Independent amplicon analyses of MIPP-Seq markedly reduce the impact of allelic dropout, amplification bias, PCR-induced, and sequencing artifacts. Using low DNA inputs of either 25 ng or 50 ng of DNA, MIPP-Seq provides sensitive and quantitative assessments of AAFs as low as 0.025% for SNVs, insertion, and deletions.ConclusionsMIPP-Seq provides an ultra-sensitive, low-cost approach for detecting and validating known and novel mutations in a highly scalable system with broad utility spanning both research and clinical diagnostic testing applications. The scalability of MIPP-Seq allows for multiplexing mutations and samples, which dramatically reduce costs of variant validation when compared to methods like ddPCR. By leveraging the power of individual analyses of multiple unique and independent reactions, MIPP-Seq can validate and precisely quantitate extremely low AAFs across multiple tissues and mutational categories including both indels and SNVs. Furthermore, using Illumina sequencing technology, MIPP-seq provides a robust method for accurate detection of novel mutations at an extremely low AAF.
Highlights
Mosaic mutations contribute to numerous human disorders
The measured Alternate allelic fraction (AAF) were linearly correlated with the expected AAFs down to 0.01% (R2 > 0.99), though as expected, individual AAFs do vary amongst individual primers (R2 > 0.98)
MIPP-Seq overcomes the limitations of powerful assays such as digital droplet Polymerase chain reaction (PCR) (ddPCR) and blocker displacement amplification (BDA) [40], which often utilize a single set of primers and probes, by using multiple unique barcoded primers for independent assessments of AAF, amplicon-specific error rates, and allelic imbalances
Summary
Mosaic mutations contribute to numerous human disorders. The identification and precise quantification of mosaic mutations is essential for a wide range of research applications, clinical diagnoses, and early detection of cancers. Traditional genetic sequencing methodologies such as whole genome (WGS) and whole exome (WES) sequencing have focused on the important contribution of germline mutations which are present in all cells throughout the human body. Postzygotic mutations, or mosaic mutations, have been heavily studied in cancers where clinical diagnostic testing of tumor and blood samples are becoming a standard practice due to improved detection sensitivities [1, 2]. In patients with focal epilepsy, somatic mutations can occur predominately in the brain region where the seizures originate and, are often undetectable using standard germline genomic analyses [3, 4, 7]. As such, improved methods for detecting and validating somatic mutations is essential for clinical testing in these patients
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