Description:
Researchers at TGen and Northern Arizona University have developed a Next Generation universal indexing amplicon sequencing strategy that can be used to amplify multiple genomic regions from a DNA sample simultaneously in a single reaction. This method uses a set of common indexed oligonucleotides with a Universal Tail (UT) sequence to quickly and efficiently add barcodes to any number of amplicons. Barcoded amplicons can then be used for Next Generation sequencing applications with no additional library preparation reagents. As this method involves an initial PCR amplification before sequencing, it doesn't require high quality double stranded DNA and can be performed on crude DNA extracts that are single stranded.
Amplicon sequencing may also be used to monitor expression levels of gene targets. RNA extracted from clinical samples would be converted to cDNA and used as template for the multiplex amplicon sequencing assay. Differential numbers of reads aligning to the amplicon reference sequences would correspond to different target copy numbers in the RNA sample.
High throughput amplicon sequencing of genomic regions of interest can be very useful for a variety of molecular genetic genotyping applications, including forensic and clinical sample analysis. Next Generation sequencing platforms provide for rapid, high yield sequence data, enabling the sequencing of multiple amplicons from many samples in a short period of time. However, efficient use of high-throughput sequencing platforms requires the use of sample barcoding, which can be cumbersome and expensive when dealing with dozens to hundreds or thousands of samples, and can be further compounded if multiple amplicons are to be sequenced from each sample. The use of UT sequences allow for multiplex preparation of amplicons from a common set of indexing oligo sequences.
Link to Issued US Patent 10,550,427
