Rare Event Detection Using Error-corrected DNA and RNA Sequencing

This method can help answer some key questions in various fields of research, such as detection of precancerous clones, prognostic assessment of leukemia patients following treatment, and identification of potentially pathogenic mutations in bone marrow donors. Demonstrating this procedure with me will be Spencer, he is a technician in the lab. This video describes how to combine the Error-corrected Sequencing Protocol with Illumina chemistry and commercially-available gene panels to detect clonal single-nucleotide variants and small indels at at one in 10, 000 sensitivity.

To incorporate the required unique molecular identifiers, customized 16N i5 and i7 adapters use PCR. First, prepare the Q5 Master Mix, which uses a polymerase with higher fidelity than the commercially available alternative. Then, for each reaction, combine 37.5 microliters of Q5 Master Mix, six microliters of five micromolar 16N i5 adapters, which are the unique molecular identifiers, and six microliters of of i7 adapters.

If the goal is multiplexing, then use different i7 adapters in separate samples. Now, run the PCR using the following parameters:thirty seconds at 98 degrees Celsius, followed by four to six cycles of ten seconds at 98 degrees Celsius, thirty seconds at 66 degrees Celsius, and thirty seconds at 72 degrees Celsius. Finish the reaction with a two minute extension at 72 degrees Celsius and hold at four degrees Celsius.

Then the both PCR cycles you have to do on them most likely depends on the size of the gene panel that you use, from our experience, a four cycle PCR is sufficient if the gene panel has about 1500 different paths of gene-specific oligos, whereas a panel with about five to 600 pairs of oligos requires about six cycles of PCR. Next, clean up the PCR reactions using magnetic beads, add 56.25 microliters of magnetic bead solution into each 75 microliter PCR product, then transfer each reaction to a separate 1.5 milliliter low-binding tube, mixed by pipetting up and down at least ten times, and wait five minutes. Next, transfer the mixes to a magnetic holder, and let the supernatants clear for two minutes.

Then, remove and discard the supernatants. Next, add 200 microliters of 70%ethanol, wait thirty seconds, and then remove the ethanol. Repeat this ethanol wash step once, and then let the beads air dry for five minutes.

Finally, elute the modified libraries from the beads in twenty microliters of double distilled water. Then, place the tubes onto a magnetic rack to separate the magnetic beads from the eluent. First, make ten-fold serial dilutions of the ECS libraries from the previous step, down to one part per 1, 000 in PCR strips.

Next, prepare a Master Mix in a 1.5 milliliter tube. For each reaction, combine 10 microliters of ddPCR EvaGreen Mix, 0.2 microliters of P5 primer, 0.2 microliters of P7 primer, and 4.6 microliters of double distilled water. Then, pipet 15 microliters of the EvaGreen Master Mix into a new set of tubes, and finally add five microliters of one to 1, 000 ECS cleaned up product to the Master Mix.

Next, make PCR droplets using the droplet generator. First, load the cassette, pipet 70 microliters of droplet generation oil into the well of the cassette, labeled oil, and pipet 20 microliters of the ddPCR reaction mixture into the well labeled sample. Then, cover the cassette with a rubber gasket, and load it into the droplet generator, and all the droplet generation to proceed.

Now, using a multi-channel pipet, slowly, over five seconds, load 45 microliters of droplets taken from each cassette well, onto a new PCR plate. Then, seal the PCR plate with an alluminum foil. Now, amplify the signal in the droplets using the following conditions:five minutes at 95 degrees Celsius, followed by 40 cycles of thirty seconds at 95 degrees Celsius, and one minute at 63 degrees Celsius, then, cool the reaction to four degrees Celsius for five minutes before raising it to 90 degrees Celsius for five minutes, and then holding at four degrees Celsius.

Next, prepare the ddPCR template droplet reader machine. Select the parameters for absolute quantification, and the use of the QX200 ddPCR EvaGreen Supermix. Then, run the reactions through the droplet reader.

When the ddPCR analysis is complete, apply the same divisive thresholds to each sample. Then, normalize each library to the desired number of molecules. Begin with making 50 microliter reactions, consisting of 25 microliters of Q5 Master Mix, two microliters of one micromolar P5 primer, two microliters of one micromolar P7 primer, and 21 microliters of normalized DNA library.

Next, run the PCR using the following parameters:30 seconds at 98 degrees Celsius, followed by 16 cycles of 10 seconds at 98 degrees Celsius, thirty seconds at 66 degrees Celsius, and thirty seconds at 72 degrees Celsius, then two minutes at 72 degrees Celsius, and hold at four degrees Celsius. Next, quantify the concentration of DNA in the libraries, and pool the libraries in equal molar amounts for sequencing, target roughly four nanomolar. Now, sequence the pooled ECS library using the Illumina sequencing platform, with the following sequencing settings:two times 144 paired-end reads, eight cycles of index one, and 16 cycles of index two.

The DNA of a patient with a mutation in GATA1, was diluted in commercial genomic DNA at the original VAF of 0.19. Using the described protocol, ECS proved to be quantitative to a level of one to 10, 000 for single nucleotide variant. Next, buffy coat samples from twenty healthy individuals were analyzed using a commercial sequencing panel that consists of 568 amplicons.

In summary, 109 clonal somatic mutations were present in both replicates of at least one collection time point with variant allele fractions ranging from 0.0003 to 0.1451.21 of the mutations with known cosmic representations were selected and each was validated using digital droplet PCR. To demonstrate the protocol's error-corrected expression level, a customized gene panel consisting of 415 genes known to be associated with various cancers, was used to produce a library built from the most commonly express exon of each gene. The expression level of low-abundance transcript was highly reproducible between replicates.

Digital droplet PCR was then used to validate six selected genes with varying expression. A comparison shows that the expression level of genes was correctly captured by the ECS protocol, without the need for normalization. Once you have mastered the technique, this technique can be done comfortably in one and a half days.

Bulk of the time will be incubation, and manual handling takes up about 30-40%of the total time required, depending on how many samples the researchers are processing at the same time. While attempting this procedure, it is very important to have a replicate sequencing library for the same sample. This will give you more confidence that a low-frequency mutations is true positive if the mutation itself is independently core in both replicates.

Following this procedure, other matters such as ECS RNA could be performed in order to answer questions such as detection of low-abundance transcript, or fusion transcript. After it's development, this technique paved the way for researchers primarily in the field of hematological malignancies, and we use this technique to explore pre-leukemic clones in healthy individuals as well as to detect minimal-residual disease in leukemia patients who are in remission.