The new progress of the liquid biopsy technology

High-throughput sequencing of Circulating tumor DNA (ctDNA) is expected to achieve a personalized cancer treatment. However, the cell-free DNA (cfDNA) in blood is limited, limiting the analytical sensitivity. To this end, researchers at Stanford University have developed a method for correcting errors can be detected frequencies as low as 0.004% of the mutant allele.

In the study published in the journal Nature Biotechnology on Monday, the researchers introduced the approach called integrated digital error suppression (IDES). It is based on the Stanford team developed before a ctDNA detection technology called CAPP-seq, and it has been acquired by Roche.

Previously, the limit of detection CAPP-seq technology is 0.02%, but many have the wrong sequencing fragments. To resolve these errors, the research team first devised a molecular barcodes strategy. Many ctDNA detection developers also use this program to reduce the error rate. Single-stranded DNA and double-stranded DNA barcodes strategies exist, but there are drawbacks. Double-stranded DNA barcodes in reducing errors on better, but less efficient than single-stranded DNA barcoding is therefore not suitable ctDNA limited amount of samples.

To this end, they set out to design a hybrid strategy. First, they devised sequencing connector that can be used for molecular barcodes of single and double stranded. Each strand of the double-stranded molecule of the first four-base mark a bar code, bar code called an index. Then they add two dinucleotide barcode on each of the two strands of the joint, called an insertion bar code. After sequencing, the bar code can be inserted into a complementary match to reconstruct the original double-stranded DNA molecule.

The second step, the Stanford team designed a computational tool that can correct sequencing or PCR system error. To achieve this, they first samples of 12 healthy adults carry CAPP-seq detection. The researchers reported that, despite all kinds of SNV has a background error, but the most common is a G → T transversion, and the error C → T and G → A also has.

They found that, G → T error occurs because the Hybrid Capture Process oxidative damage. They used a computational method to suppress these errors. The combination of these two strategies, so that the error rate has dropped by 15 times, which is by 30 healthy control samples and 142 samples of non-small cell lung cancer confirmed.

Researchers are also NSCLS samples on this verification test. First, they examined 41 patients with advanced NSCLS EGFR mutation hotspot. They detected 88 plasma samples to 412 EGFR mutation, all variations have been confirmed by a tumor biopsy sample. In addition, this test did not check out any false positives.

They then assessed the technical limitations in the detection of the reference cell lines. They created a reference cell lines mixture, wherein the frequency of allelic variation at 0.05-1.6%. They found that the bar code policy and calculation methods are complementary correction, combined with better results. Theoretical detection limit of this method is 0.00025%.

Finally, they used this method to monitor the 30 NSCLC patients with mutations; these patients have had tumor genotyping. The researchers found that they could detect the mutation frequency as low as 0.004%. "To our knowledge, this is the minimum amount of ctDNA with deep sequencing detection by far," the authors wrote.

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