The overall goal of this procedure is to identify somatic mutations in cancer associated genes of cells from tumor tissue by using barcoded multiplexed, DNA libraries, followed by massively parallel sequencing. This is accomplished by first ligating barcoded sequencing adapters to DNA fragments isolated from preserved tumors. The second step of the procedure is hybridization to custom biotinylated oligonucleotides, complementary to all the protein coating exons of 279 oncogenes and tumor suppressor genes.
The third step is to perform massively parallel sequencing of the barcoded pools from the captured libraries. The final step is to search the sequence data for cancer associated alterations for the 279 targeted genes. This procedure can reveal sequence mutations, small insertions, small deletions, copy number alterations, and select structural alterations.
So the main advantages of this technique over existing methods are that it allows one to investigate entire exons for both the common and rare mutations, as well as identify additional genomic alterations such as copy number losses and gains, and attain greater sensitivity in heterogeneous Samples. This method can help answer key questions in the cancer genomics field, such as what are the key driver mutations in various cancer types, and do these mutations in clinical specimens correlate with outcomes or response to targeted therapies? The implications of this technique extends toward diagnosis and treatment of cancer because mutations may be identified prospectively and be used to help guide patients to appropriate therapies and clinical trials.
Visual demonstration of this method is critical as a bee clean step is difficult to learn due to a caution technique needed while washing and eluding DNA off beads Prepare the end repair master mix. Mix the appropriate volumes to have 50 microliters per sample. Aliquot the 50 microliters of each sheer DNA into separate wells of an ID six well plate.
Then add 50 microliters of the prepared end repair master mix to each reaction and incubate the plate in a thermocycler for 30 minutes at 20 degrees Celsius to clean up the reaction. First, add 200 microliters of AMP pure XP beads to each sample and gently pipette the entire volume up and down 10 times using a multi-channel pipetter. Follow this by incubating the plate at room temperature for 15 minutes.
Next, move the plates to the magnetic stand and allow them to clear for 15 minutes at room temperature. Once cleared, gently remove and discard most of the supernatant taking care not to disturb the beads. Some liquid may remain in the wells.
Then gently add 200 microliters of freshly prepared 80%ethanol to each sample and incubate the plate at room temperature for 30 seconds. Carefully pipette away the ethanol and repeat the wash once more. Next, let the plate dry at room temperature until all the alcohol escapes.
Now resuspend the dried beads in 44.5 microliters of sterile water. Gently flow each sample through the pipette 10 times with the last ejection. Rinse down any beads attached to the side of the well.
Now incubate the water suspended beads at room temperature for two minutes, and then move them to the magnetic stand for five minutes until the wells are clear. Lastly, gently transfer 42 microliters of the clear supernatant in each well, which contains the sample to a new well. The samples can now be stored at minus 20 degrees Celsius for up to a week.
Begin by preparing the D tailing master mix in a sterile micro centrifuge tube. Add eight microliters of the prepared DA tailing master mix to each well of end repaired DNA. Then incubate the plate in a thermocycler for 30 minutes at 37 degrees Celsius to clean up the reaction.
Use the same process outlined in the previous section at twice the volume and finish by Resus suspending the beads in 33.75 microliters of sterile water. At this point, the samples can then be stored at minus 20 degrees Celsius for up to a week. To continue prepare a ligation master mix for 15 microliters per reaction.
Add 15 microliters of the ligation master mix to each well containing the D tail DNA. Next, add 1.25 microliters of the appropriate 25 micromolar. Next flex barcoded adapter.
To each, well, each sample should receive a separate barcoded adapter. Once the adapters are loaded, incubate the plate for 15 minutes at 20 degrees Celsius. Now using 50 microliters of beads, perform a post adapter ligation cleanup and resuspend the sample in sterile water to 50 microliters.
Then do the cleanup process a second time finishing with 23 microliters of water. At this point, the samples can then be stored at minus 20 degrees Celsius for up to a week. The next step is to library amplify each sample, which is detailed in the text protocol on ice thaw, the universal and index oligonucleotide blockers, the CCAP easy library, and the nimble gen capture components, which include the con TNA two X hybridization buffer, and hybridization component.
A.Now prepare a one millimolar pool of index oligonucleotide blockers corresponding to the specific barcoded adapter sequences used in the library preparation. Combine one microliter of each blocker and vortex them together for 10 seconds In a new 1.5 milliliter tube, prepare the capture mix consisting of five microliters of conte A at one milligram per milliliter, two microliters of universal blocker and two microliters of the blocker pool. Pool 24 barcoded libraries into a single reaction.
Add a total of one to three micrograms of pooled barcoded sequence library to the capture mix. Punch 15 to 20 holes in the cap using a 20 gauge or smaller needle speed vac the mixture in A DNA vacuum concentrator at 60 degrees Celsius until it is completely dry. Next, rehydrate the mix in hybridization buffer and hybridization component A.Cover the holes in the cap with tape and vortex the sample for 10 seconds.
Then centrifuge the mix at maximum speed for 10 seconds. Now, briefly incubate the mix at 95 degrees Celsius to denature the DNA. Follow this with 10 seconds of centrifugation at maximum speed at room temperature in a 0.2 milliliter PCR strip tube mix 2.25 microliters of ccap easy library capture probes to the same volume of sterile nuclease free water.
Then at the centrifuge reaction mix to the tube and gently flow the total volume through a pipette tip 10 times. Now incubate the 0.2 milliliter tube in a thermal cycler at 47 degrees Celsius for 48 to 96 hours. Maintain the thermal cycler's lid temperature at 57 degrees Celsius to prevent evaporation and stabilize the reaction temperature days later.
Use the steps for wash and recovery of the captured DNA. Then use a modified Roche nimble gen protocol to amplify the captured DNA finish by quantifying the amplified captured DNA using the qubit high sensitivity assay. One pool of 24 barcoded sequence libraries that contained 12 tumor normal pairs was captured using probes of 279 cancer genes and sequenced as two by 75.
Base pair reads on a single lane of a HighSeq 2000 flow cell tumor and normal libraries were pooled in a two to one ratio. The IGV image shows specificity of sequence coverage at exons of EGFR in a lung cancer gray bars represent unique sequence reads. The heterozygous T two G mutation on the right was present in 24%of the reads, indicating that it is a somatic L 8 58 R amino acid substitution.
None of the reads of normal lung tissue showed this TTA G mutation, some indels in a colorectal cancer tumor, normal pair showed a somatic frameshift insertion in a PC or a somatic frameshift deletion of seven base pairs. In TP 53, copy number alterations were also seen between tumor normal pairs. Each data point represents a single exon from 279 target genes.
Copy number gains and losses are inferred from increases and decreases in tumor sequence Coverage Once mastered, this technique can be done in six and a half hours for sharing library preparation and 48 to 96 hours for capture hybridization if performed properly. While performing this assay is extremely important to be cautious of contaminants during library preparation, as it may confound data analysis Following this procedure, other methods like Sanger sequencing and fragment analysis can be performed in order to answer questions like, how do I validate a questionable alteration? After watching this video, you should have a good understanding of how to prepare barcoded DNA libraries and perform Exxon capture on these pooled libraries.
