The overall goal of this next generation sequencing targeted Amplicon Assay is to detect pathogenic mutations in patient tumors for therapeutic use, including diagnostic, prognostic, and response to targeted therapy. This method answers key questions, such as whether a patient has a mutation in a gene that can benefit from a targeted or alternative therapy. The main advantage to this technique is that actionable mutations, both common and rare, across many genes can be detected in any tumor tissue using a single assay.
Because the techniques and the technology are so complex and the steps are so difficult to learn, it is critical to demonstrate this method visually. This is a defining moment in advancing Medical Oncology. With the adoption of next generation sequencing into the clinic, we will have more and better cancer treatments for our patients.
Our CPD Technologists, Barentt Li, Patrick Candrea, Karthik Ganapathy, and Joe Grubb will demonstrate this procedure. I will also aid in this process to show the more complex parts of the assay. After extracting genomic DNA according to the text protocol, following the manufacturer's protocol, run two microliters of the extracted DNA on a fluorometer to obtain the working concentration of the sample.
To create an Amplicon Library, in a semi-skirted 96-well plate called the Hyb Plate, add the necessary volume of low EDTA TE, five mircoliters of the panel's Oligo Tube, add 100 to 250 nanograms of genomic DNA to each corresponding well. This is the single most vital step, as any mix up here will have severe consequences. Following the addition of the Oligo Hybridization for Sequencing Reagent 1, and spinning according to the text protocol, incubate the Hyb Plate in the preheated hybridization incubator at 95 degrees Celsius for one minute.
After slowly cooling the incubator to 40 degrees Celsius, remove the plate from the incubator, and transfer the entire volume of each sample from the Hyb Plate to the center of the previously prepared corresponding, prewashed filter plate unit, or FPU. Cover the FPU, and centrifuge at 2, 250 times G and 20 degrees Celsius of three minutes. Then, add 45 microliters of SW1, and centrifuge again.
After a second wash with SW1 and a wash with UB1, add 45 microliters of Extension Ligation Mix 3 to each sample well, and pipe it up and down three times to mix. Use adhesive aluminum foil to seal the plate, and incubate the entire FPU assembly in a preheated 37 degree Celsius incubator for 45 minutes. To index the PCR Amplification, arrange the primers in the Index Amplification Plate, or IAP Fixture, and aliquot the indexes being used into the corresponding wells in the plate, then add 22 microliters of the PCR Master Mix, 2E12, and pipe it up and down three times.
Next after spinning down the 45 minute Extension Ligation Reaction according to the text protocol, add 25 microliters of 50 millimolar sodium hydroxide to each sample well of the FPU, and pipe it up and down at least six times, ensuring that the pipette tip comes in contact with the membrane. Then with the plate slightly tilted and a multi-channel pipette set to 20 microliters, pipette the sodium hydroxide in the FPU plate up and down at least six times. Transfer the elution from the FPU to the corresponding column of the IAP.
Gently pipette up and down to thoroughly combine the DNA with the PCR Master Mix before running the PCR program outlined in the text protocol. After verifying the library yield and incubating the samples with Magnetic Purification Beads according to the text protocol, add 30 microliters of EBT to each well of washed beads. Pipette up and down a few times to ensure the beads come off the side of the tube.
Once the plate has been incubated with and then without shaking, place the plate on the magnetic stand and transfer 20 microliters of the supernatant to a whole new plate called the Library Normalization Plate, or LNP. Following the preparation of the normalization mix, with intermittent inversion, and vortexing of the solution, add 45 microliters to each sample of the LNP. Then, use clear adhesive film to seal the plate, and shake at 1, 800 RPM for 30 minutes.
After washing the beads, remove the LNP from the magnetic stand and to elute the sample, add 30 microliters of fresh 0.1 normal sodium hydroxide to each well. Then, shake the LNP at 1, 800 RMP for five minutes. Place the LNP back on the magnetic stand, and after the supernatant has cleared, transfer 30 microliters of the elution to previously prepared Library Normalization Storage Buffer 1 in the Storage Plate.
Bend down the plate, then add five microliters of each sample to be sequenced to a labeled Pooled Amplicon Library, or PAL, 1.5 milliliter tube. Depending on what sequencing chemistry is being used, add four to 10 microliters of the PAL to 590 to 596 microliters of Buffer HT1. Clean and load the Flow Cell.
Load the reagents. And, follow the on-screen prompts to start the sequencing run. After the sequencing run has completed, execute the Bioinfomatics Pipeline.
Analyze the run statistics to ensure the sequenced library has passed the lab-determined quality control metrics. Finally, in a Genomic Data Viewer, manually review each variant by viewing the BAM files. This is a summary of the most important run statistics, not including the mean coverage, used to determine if a library prep sample has passed QC.As seen here for Case One, Bioinformatics detected four reportable AML-associated mutations.
A missense mustation in FLT3, a missense mutation in IDH2, and a frameshift mutation in NPM1. Mutations in FLT3 are observed in about 25%of adult patients with AML. The prognostic significance of FLT3 Kinase Domain Point Mutations, as seen in this AML patient, has an unclear impact on prognosis.
Isocitrate Dehydrogenase 2, or IDH2, encodes an epigenetic modifier that is commonly mutated in AML. Mutations in the gene for Nucleophosmin, or NPM1, are one of the most commonly acquired mutations in AML. This table lists all the exonic variants for Case Two.
Two disease-associated mutations were detected. An in-frame insertion in Exon 20 of ERBB2, and a missense mutation in TP53. HER2/neu, or ERBB2, encodes a Tyrosine Kinase Receptor.
Activating HER2/neu Exon 20 insertions are observed in two to 4%of Lung Adenocarcinomas and account for the majority HER2/neu mutations observed in lung cancer. TP53 changes are also common in cancer. It is a necessary to pay close attention to the quality and the quantity of extracted DNA.
This is especially important for FFPE samples. We try often highly degraded with a variable the DNA yield. During the validation process of the clinical assay, metrics for DNA quality and quantity should be established to each sample before advancing the DNA into library preparation.
In addition to the library preparation, it is critical to validate a Bioinfomatic Spy Plan, by determining cutoff values for the sequencing run, quality control statistics, library preparation statistics, depth of courage of an Amplicon, and the minimum reportable allele frequency. Once mastered, the library prep can be performed in a working day. But, the overall assay workflow requires more time for DNA extraction, sequencing, data processing, and variant review.
This procedure is designed to only look at the genomic DNA mutations in certain key hot spots. Other masses that use RNA can be performed to answer additional questions like a differential expression of genes, associate the risk tumors, and the structural rearrangements. Major advancements are on the horizon.
The adoption of automation and the incorporation of a unique molecular barcoding will allow laboratories to decrease turnaround time, use less sample input, and detect variants at a very low allele frequency. Detection of disease-associated mutations in cancer specimens has been standard of care for decades. NGS is a less biased approach to sequencing multiple genes associated with many cancers in parallel, leading to the identification of multiple mutations and better treatments for patients.
Thank you for watching, and good luck with your experiments.
