capp-seq is reported to be 2.5 molecules in 1,000,000. tagged amplicon deep sequencing (tam-seq) tam-seq allows targeted sequencing of entire genes to detect mutations in ctdna. first a general amplification step is performed using primers that span the entire gene of interest in 150-200bp sections. then, a microfluidics system is used to attached adaptors with a unique identifier to each amplicon to further amplify the dna in parallel singleplex reactions. this technique was shown to successfully identify mutations scattered in the tp53 tumor suppressor gene in advanced ovarian cancer patients. the sensitivity of this technique is 1 in 50. safe-sequencing (safe-seq) this method was originally described by bert vogelstein and his group at johns hopkins university. safe-seq decreases the error rate of massively parallel sequencing in order to increase the sensitivity to rare mutants. it achieves this by addition of a unique identifier (uid) sequence to each dna template. the dna is then amplified using the added uids and sequenced. all dna molecules with the same uid (a uid family) should have the same reported dna sequence since they were amplified from one molecule. however, mutations can be introduced through amplification, or incorrect base assignments may be called in the sequencing and analysis steps. the presence of the uid allows these methodology errors to be separated from true mutations of the ctdna. a mutation is considered a ‘supermutant’ if 95% of the sequenced reads are in agreement. the sensitivity of this approach is 9 in 1 million. duplex sequencing this method is an improvement on the single uids added in the safe-seq technique. in duplex sequencing, randomized double-stranded dna act as unique tags and are attached to an invariant spacer. tags are attached to both ends of a dna fragment (α and β tags), which results in two unique templates for pcr - one strand with an α tag on the 5’ end and a β tag on the 3’ end and the other strand with a β tag on the 5’ end and an α tag on the 3’ end. these dna fragments are then amplified with primers against the invariant sequences of the tags. the amplified dna is sequenced and analyzed. dna with the duplex adaptors are compared and mutations are only accepted if there is a consensus between both strands. this method takes into account both errors from sequencing and errors from early stage pcr amplification. the sensitivity of the approach to discovering mutants is 1 in 10^7. integrated digital error suppression (ides)-enhanced capp-seq ides improves capp-seq analysis of ctdna in order to decrease error and therefore increase sensitivity of detection. reported in 2016, ides combines capp-seq with duplex barcoding sequencing technology and with a computational algorithm that removes stereotypical errors associated with the capp-seq hybridization step. the method also integrates duplex sequencing where possible, and includes methods for more efficient duplex recovery from cell free dna. the sensitivity of this improved version of capp-seq is 4 in 100,000 copies. whole-genome sequencing whole-genome sequencing investigations have been performed on ctdna present in different patients with treatment-resistant prostate cancer (the vast majority, and in some cases, metastatic), bladder cancer and control patients who did not present this dna, including somatic mutations and structural rearrangements in their genomes. this novel and promising technique has provided information on resistance to treatment with androgen receptor signaling inhibitors, intratumoral heterogeneity (thanks to phylogenetic evolution and molecular chronology), chromosomal instability, contribution of ctdna to metastasis through global transcriptomic patterns (taking into account nucleosomes present in transcription start sites (tsss) and ar-binding sites (arbs). in this way, the genomic and transcriptomic evolution of ctdna can be observed, performed in living patients who are developing resistance to treatment, therefore, ctdna sequencing is elementary to identify clinically relevant differences in the cancer phenotype and to see how therapy is affecting patients. furthermore, the relative homogeneity in driver gene alterations among metastases justifies that genomic and functional alterations in prostate cancer are shared between ctdna and tissue. this makes ctdna a powerful emerging tool for the detection of genetic mutations at the genomic scale in patients suffering from metastatic cancer to observe the clinical relevance of the clonal composition of these tumors to understand better cancer control. this subclonal reconstruction based on ctdna thanks to whole-genome sequencing poses a unique set of challenges and opportunities for scientific research in oncology. furthermore, serial ctdna reveals treatment-driven selection for androgen receptor augmentation because it increases the dimensionality of the data. further work is needed to understand how metastatic location and size, in relation to tumor burden, influence circulating tumor dna and the choice of new techniques to select other lesions that reflect clinically dominant disease. considerations “normal” vs tumor dna detection one of the challenges in using ctdna as a cancer biomarker is whether ctdna can be distinguished with cfdna from normal cells. cfdna is released by non-malignant cells during normal cellular turnover, but also during procedures such as surgery, radiotherapy, or chemotherapy. it is thought that leukocytes are the primary contributors to cfdna in serum. research ctdna in cancer screening the clinical utility of ctdna for the detection of primary disease is in part limited by the sensitivity of current technology to detect small tumors with low levels of ctdna present and a priori unknown somatic mutations. ctdna in cancer monitoring evidence of disease by traditional imaging methods, such as ct, pet or mri may be absent after tumor resection. therefore, ctdna analysis poses a potential avenue to detect minimal residual disease (mrd), and thus the possibility of tumor recurrence, in cases where bulk tumors are absent by conventional imaging methods. a comparison of mrd detection by ct imaging compared to ctdna has been previously done in individuals with stage ii colon cancer; in this study, researchers were able to detect ctdna in individuals who showed no sign of clinical malignancy by a ct scan, suggesting that ctdna detection has greater sensitivity to assess mrd. however, the authors acknowledge that ctdna analysis is not without limitations; plasma samples collected post-operatively were only able to predict recurrence at 36 months in 48% of cases. ctdna as a prognostic biomarker the question of whether measurement of the amount or qualities of ctdna could be used to determine outcomes in people with cancer has been a subject of study. as of 2015 this was very uncertain. although some studies have shown a trend of higher ctdna levels in people with high stage metastatic cancer, ctdna burden does not always correlate with traditional cancer staging. as of 2013 it appeared unlikely that ctdna would be of clinical utility as a sole predictor of prognosis. cancer research the emergence of drug-resistant tumors due to intra- and inter-tumoral heterogeneity an issue in treatment efficacy. a minor genetic clone within the tumor can expand after treatment if it carries a drug-resistant mutation. initial biopsies can miss these clones due to low frequency or spatial separation of cells within the tumor. for example, since a biopsy only samples a small part of the tumor, clones that resides in a different location may go unnoticed. this can mislead research that focuses on studying the role of tumor heterogeneity in cancer progression and relapse. the use of ctdna in research can alleviate these concerns because it could provide a more representative 'screenshot' of the genetic diversity of cancer at both primary and metastatic sites. for example, ctdna has been shown to be useful in studying the clonal evolution of a patient’s cancer before and after treatment regimens. early detection of cancer is still challenging but recent progress in the analysis of the epigenetic features of cfdna, or the fragmentation pattern unlock improve the sensitivity of liquid biopsy. furthermore, ctdna analysis is an emerging tool for understanding the clonal composition of metastatic tumors, detecting different mutations on a genomic scale, studying subclonal diversity that affects the prognosis of the disease as different resistant phenotypes can be found and the appearance of new mechanisms of genomic and transcriptomic resistance to treatment. challenges for implementation implementation of ctdna in clinical practice is largely hindered by the lack of standardized methods for ctdna processing and analysis. standardization of methods for sample collection (including time of collection), downstream processing (dna extraction and amplification), quantification and validation must be established before ctdna analysis can become a routine clinical assay. furthermore, creation of a panel of ‘standard’ tumor-associated biomarkers may be necessary given the resolution of current ctdna sequencing and detection methods. sequencing tumor-specific aberrations from plasma samples may also help exclude contaminating cfdna from analysis; elevated levels of cfdna from normal cells may be attributed to non-cancer related causes. these sequencing techniques can also determine the clonal evolution of cancer, tumor heterogeneity and drug resistance mechanisms involved in cancer.