Amplicon sequencing, which relies on PCR-amplification of the targeted regions of the genome using sequence-specific primers and probes, is one the most common NGS-based method used in target enrichment which has many applications in clinical and industrial fields. It is considered a more suitable option for efficient gene panel testing and in production-scale applications such as industrial genomic screening and clinical diagnostics. Its common applications in the clinical field include inherited disease testing, cancer research, pathogen detection, and pharmacogenomics testing.
Tagged amplicon deep sequencing, or TAM-Seq, enables the amplification of the entire genome by tilling short amplicons through the usage of two-step amplification. It produces libraries tagged with the specific barcodes of the samples. TAM-seq allows targeted sequencing of the entire genome to detect mutations in circulating tumor deoxyribonucleic acid (ctDNA). It can also be scrutinized for genetic alterations without background or prior knowledge of the tumor’s driver mutations.
In TAM-seq, the primers that span the entire gene of interest (about 150-200 base pairs) are initially used to bind to the template during the pre-amplification process to amplify the original signal. This step was done to be able to control sampling errors, as well as allelic loss. And then, the templates undergo an individual amplification process for purification. In the second step, a microfluidics system is utilized to attach adaptors with a unique identifier to each amplicon. Again, this is to further amplify the DNA in parallel singleplex reactions. The advantage of using this two-step amplification is that TAM-seq can identify mutations ~ 2% MAF with a sensitivity of more than 97%. TAM-seq allows the detection of cancer-specific mutations down to allele frequencies as low as two percent. Also, it has been reported to detect known hotspots in EGFR and TP53 tumor suppressor gene in advanced ovarian cancer patients as low as 0.2% or 1 in 50.
High-grade Serous Ovarian Cancer
In 2012, tagged-amplicon deep sequencing enabled the interrogation of six genes across a large region of the genome (about 5995 bases) to detect low-frequency mutations in cell-free DNA. The study revealed that TAM-seq technology has 97% sensitivity, as well as specificity for detection of mutations at two percent fraction. And that it was also able to identify mutations down to 0.14% allele fraction. The study concluded that TAM-seq can assay multiple mutations in parallel to monitor tumor dynamics. Using this technology, it is also possible to identify de novo mutations direct from the circulating free DNA of the patient and identify the origin of metastatic relapse.
TAM-sequencing was also used for the detection of both abundant and rare mutations in the circulating DNA, extracted from the blood plasma, of ovarian and breast cancer patients. This specific sequencing technique allowed the researchers to monitor changes in tumor burden by sampling only patient plasma over time. It was concluded that this approach, combined with faster and more accurate amplification strategies, could potentially be used for personalized medicine.
Pancreatic Ductal Adenocarcinoma (PDAC)
TAM- deep sequencing has been also used to detect ctDNA in PDAC patients. It has been utilized to interrogate tumor variants across broad genomic regions which include many cancer-associated target genes. This is through the extraction of blood samples from patients with various cancer types. It was found out that TAM-sequencing is indeed helpful in facilitating evaluations of tumor burden, intra-tumor genetic heterogeneity, clonal expansion during the disease progression stage, as well as in the emergence of resistant mutations.