Ensemble of nucleic acid absolute quantitation modules for copy number variation detection and RNA profiling.
basic_science · Level V
Where this comes from
- Record sourced from PubMed, PMID 35379811.
- Also identified by DOI 10.1038/s41467-022-29487-y and PMC identifier 8979981.
- Licence recorded as CC BY.
- The licence permits redistribution, so the abstract is shown in full and the full text is available from the publisher.
Abstract
Current gold standard for absolute quantitation of a specific DNA sequence is droplet digital PCR (ddPCR), which has been applied to copy number variation (CNV) detection. However, the number of quantitation modules in ddPCR is limited by fluorescence channels, which thus limits the CNV sensitivity due to sampling error following Poisson distribution. Here we develop a PCR-based molecular barcoding NGS approach, quantitative amplicon sequencing (QASeq), for accurate absolute quantitation scalable to over 200 quantitation modules. By attaching barcodes to individual target molecules with high efficiency, 2-plex QASeq exhibits higher and more consistent conversion yield than ddPCR in absolute molecule count quantitation. Multiplexed QASeq improves CNV sensitivity allowing confident distinguishment of 2.05 ploidy from normal 2.00 ploidy. We apply multiplexed QASeq to serial longitudinal plasma cfDNA samples from patients with metastatic ERBB2+ (HER2+ ) breast cancer seeking association with tumor progression. We further show an RNA QASeq panel for targeted expression profiling.
Medical subject headings
- Breast Neoplasms
- Cell-Free Nucleic Acids