Oncology research is pushing the bounds of early cancer detection and monitoring. Cells shed degraded DNA into the bloodstream due to normal processes. Cell free or circulating DNA can be isolated from a patient’s plasma to assay for circulating tumor DNA released from tumors. Researchers are developing tools for the early detection and monitoring of ctDNA to improve patient care.
Like many diseases, cancer treatment works better and survival rates increase the earlier the cancer is detected. Traditional methods of tumor detection, such as MRI and mammograms, are reliable, but typically require patients to be in an advanced stage of cancer development. Blood screening, also called liquid biopsy, is an advanced method of early detection that uses circulating tumor DNA (ctDNA) to directly measure cancer DNA. Tumors that are too small to detect manually shed nucleic acids, also called cell-free DNA (cfDNA), into the patient’s blood. Genetic signatures from the tumor can be detected through next generation sequencing (NGS) of the DNA extracted from the patient’s blood. Deep sequencing of this DNA enables detection of cancer mutations at low levels in the blood. Since the tumors are still very small, liquid biopsy enables asymptomatic screening before the patient has cancer symptoms and is a noninvasive testing method of finding cancer at the early stages.
To prevent patient discomfort, the blood taken from a patient to perform liquid biopsy is very small. Consequently, the amount of cfDNA on which to perform sequencing is limited. Sequencing technologies must be extremely efficient to take advantage of the precious samples. Next generation sequencing has 3 main steps: library prep, sequencing, and analysis. During the library preparation step, DNA is converted into libraries, which allow the sample to be sequenced. Sensitivity to tumor detection can be increased by a high conversion rate of DNA to library during the library preparation step. This allows NGS to detect low-frequency variants.
Error correction and accuracy can be further enhanced through the use of unique molecular identifiers (UMIs). UMIs are short sequences, often with degenerate bases, that incorporate a unique barcode onto each molecule within a given sample library. UMIs have been shown to reduce the rate of false-positive variant calls and increase sensitivity of variant detection. By incorporating individual barcodes on each original DNA fragment, variant alleles present in the original sample (true variants) can be distinguished from errors introduced during library preparation, target enrichment, or sequencing. Any identified errors can be removed by bioinformatics methods before final data analysis.
Early cancer screens contribute to cancer research by providing information about cancer development. As cancer is detected earlier, treatments can be developed to target mechanisms of tumor development. Early treatment targets may eventually be used to develop cancer preventions.
The same techniques used for early detection can also be used after a patient is in remission. During or after treatment, a small number of cancer cells usually remain in the patient’s blood, called minimal residual disease (MRD), and can lead to relapse. Liquid biopsy and NGS can detect minute numbers of cancer cells and determine whether the treatment has completely eradicated the cancer. MRD detected by liquid biopsy can also be used to track the efficacy of treatment and adjust the therapy the patient is receiving.
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Targeted sequencing uses deep sequencing to detect known and novel variants within your region of interest. Thus, it can be used as a method of gene expression analysis, mutation detection, and gene structure analysis.
Hybridization capture is a targeted next generation sequencing method that uses long, biotinylated oligonucleotide baits (probes) to hybridize to the regions of interest. It is particularly helpful when genotyping, detecting rare variants, and exome sequencing.
Genetic mutations are more likely to impact health outcomes when they occur in protein-coding genes. Cancer is a disease that can progress quickly, so understanding what mutations effect protein-coding genes can establish effective treatment and improve prognosis. Exons that make up the protein-coding genes comprise 1% of the human genome, so focusing sequencing on this portion of the genome is more cost-effective than whole genome sequencing and will provide more comprehensive data than other sequencing methods.
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The Lotus DNA Library Prep Kit enables streamlined preparation of high-quality next generation sequencing (NGS) libraries from double-stranded DNA (dsDNA)—generate libraries suitable for PCR-free, PCR-amplified, and targeted sequencing applications on Illumina platforms.
Ensure maximum accuracy in your NGS reads with xGen Stubby Adapter and Unique Dual Indexing (UDI) Primer Pairs. A convenient indexing option for a variety of applications from whole-genome to targeted sequencing, the kit provides adapters and unique i5 and i7 primer pairs that can be used with TruSeq™-compatible library prep for sequencing on Illumina instruments.
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