Detection of Rare Mutations in CtDNA Using Next Generation Sequencing

The overall goal of this technique is to detect mutations of low frequency in circulating tumor or ctDNA to provide clinical physicians a powerful tool to diagnose tumors and monitor tumor dynamics in response to therapy. The method of testing ctDNA can help answer key questions about what kinds of mutations a patient carries such as single nuclear-type variation, insertion deletion, copy number variation, and structural variation. The main advantage of this technqiue is high sensitivity and specificity which precise detection of low frequency mutations in ctDNA.

Demonstrating the procedures will be Yuliang Yang, a technician from my laboratory. To begin, draw 10 milliliters of peripheral blood in a collection tube and gently invert the tube up and down six to eight times to mix the contents. Store blood samples in the collection tubes at six to 37 degrees Celsius for up to 72 hours.

Centrifuge the collection tube at 1, 600 times g at room temperature for 10 minutes. Then use a disposal pipette to transfer the supernatant or plasma from the tube to four clean two milliliter appropriately labeled centrifuge tubes without disturbing the pellet. Use a clean disposal pipette to transfer one milliliter of the cells to a two milliliter appropriately labeled centrifuge tube.

Store the cells at minus 20 degrees Celsius or colder before genomic or gDNA isolation. Next, centrifuge the plasma at 1, 600 times g at four degrees Celsius for 10 minutes. Then transfer the plasma to clean centrifuge tubes properly labeled as plasma leaving approximately 0.1 milliliter of residual volume at the bottom to avoid contamination.

The buffy coat should be avoided from the plasma during the separation process. Otherwise, large DNA fragments extracted from the cells might dilute the ctDNA and affect the sensitivity of the test. Using a commercially available kit, isolate cfDNA from three milliliters of the collected plasma following the manufacturer's instructions.

Then also with a kit, isolate gDNA from 200 microliters of white blood cells following the manufacturer's instructions. To fragment the gDNA control sample by sonication, prepare one microgram of gDNA sample in 100 microliters of Tris-EDTA buffer in a clean sonication tube. Set the sonication program to 30 seconds on and 30 seconds off for 12 cycles for a total of 12 minutes.

After confirming the product contains 200 to 250 base pair fragments, transfer all fragmentation products to a new 1.5 milliliter tube containing 150 microliters of magnetic beads and incubate the sample for five minutes to select the correct fragments. Next, place the tube on a magnetic rack for 30 seconds and remove the supernatant. Then while keeping the tube on the rack, add 200 microliters of freshly prepared 80%ethanol to wash the beads.

Incubate the beads for 30 seconds before removing the supernatant and repeat the wash. Keep the tube on the rack with the lid open to air dry the beads. Then elute the DNA fragments by adding 32 microliters of 10 millimolar Tris-HCL to the beads.

To carry out the end prep reaction following the manufacturer's instructions, to a sterile nuclease-free tube, add seven microliters of reaction buffer, three microliters of the enzyme mix, 30 nanograms of cfDNA, and doubly distilled water for a total volume of 60 microliters. In a separate tube, add the same components but with one microgram of gDNA instead of cfDNA. Incubate the mixture in a thermal cycler at 20 degrees Celsius for 30 minutes followed by 65 degrees Celsius for 30 minutes without the lid heated.

Immediately following the incubation, add 30 microliters of the ligation master mix, one microliter of the ligation enhancer, and four microliters of the unique sequencing adapter to the tube. Incubate the reaction in the thermal cycler at 20 degrees Celsius for 15 minutes without the lid heated. Vortex to resuspend the magnetic beads and leave the beads at room temperature for at least 30 minutes.

Then add 87 microliters of the resuspended magnetic beads to the ligation reaction. Mix well by pipetting up and down and then incubate the sample at room temperature for five minutes. After washing and air drying the beads, elute the DNA target from the beads by adding 20 microliters of 10 millimolar Tris-HCL.

Add 20 microliters of adapter ligated DNA fragments, five microliters of index primer i7, 25 microliters of library amplification master mix, and doubly distilled water up to a total volume of 50 microliters. PCR amplify the adapter ligated cfDNA or gDNA. Then add 45 microliters of suspended magnetic beads to the PCR-enriched DNA.

Mix the sample by pipetting up and down and incubate it for five minutes. After removing the supernatant and washing the beads, use 30 microliters of 10 millimolar Tris-HCL to elute the DNA fragments with adapters. To carry out targeted DNA capture, in a sterile tube, block the sample by adding 1.5 micrograms of pooling libraries, eight microliters each of P5 block 100P and P7 block 100P, and five microliters of one microgram per microliter cock one DNA.

Dry the contents of the tube using a vacuum concentrator set at 60 degrees Celsius. Hybridize the DNA capture probes with the library by adding 8.5 microliters of 2X hybridization buffer, 2.7 microliters of hybridization enhancer, and 1.8 microliters of nuclease-free doubly distilled water. Mix by pipetting up and down and incubate in the thermal mixer at 95 degrees Celsius for 10 minutes.

Then immediately add four microliters of the custom probe. Incubate the samples in a thermal mixer at 65 degrees Celsius with the lid heated to 75 degrees Celsius for four hours. Incubate the hybridized target DNA with streptavidin beads then wash the beads to remove unbound DNA.

Use the commercial kit according to the manufacturer's instructions to get a final 20 microliters of resuspended beads with captured DNA fragments. The washing step of unbound DNA from streptavidin beads should be quick. Otherwise, the capture efficiency of the targeted DNA might be affected.

Amplify the captured DNA fragments by performing PCR using a commercial kit with two micromolar backbone oligonucleotides according to the manufacturer's instructions. Finally, add 45 microliters of suspended beads directly to the PCR product and enrich the amplified target DNA fragments bound to the beads by elution with 30 microliters of 10 millimolar Tris-HCL. Quantify the fragments and carry out sequencing and data analysis according to the text protocol.

This table outlines how ER-seq improves coverage depth by 23%compared with the traditional method. This is due to its efficient recovery of cfDNA molecules, thus greatly enhancing the analysis of rare mutations. It is also clear that the unique sequencing adapters used in ER-seq enable easy differentiation of natural and PCR-induced duplications.

As detailed in this table on the calling results, analysis based on ER-seq was 100%consistent for EGFR pL858R detection and the frequency of detection was a bit higher when compared with a traditional analysis. Importantly, ER-seq analysis enabled the detection of other relatively low frequency mutations including EGFR pT790M which was not recognized by traditional analysis due to high background noise. Once mastered, this technique can be done in 48 hours if it is performed properly.

While attempting this procedure, it's important to remember to avoid loss of a very low quantity of cfDNA during extraction, library prep, and bead wash after hybridization. After watching this video, you should have a good understanding of how ctDNA from patient was collected, prepared, and targeted based on unique identified for sequencing. After its development, this technique paved the way for the researchers in the field of liquid biopsy to explore better practices in making clinic decisions.