VDJ-Seq: Deep Sequencing Analysis of Rearranged Immunoglobulin Heavy Chain Gene to Reveal Clonal Evolution Patterns of B Cell Lymphoma

The overall goal of this procedure is to characterize the clonal composition of lymphoma samples for the identification of clonal evolution patterns of lymphoma relapse. This method can help address key questions in the lymphoma field, such as how relapsed tumors origin from the original tumors. The main advantage of this protocol is that it adapts the latest high throughput sequencing technology to allow a more comprehensive mapping of lymphoma commonalities.

In this protocol, DNA will be extracted from thin sections of frozen OCT embedded as well as formal and fixed paraffin embedded normal or malignant tissue to extract DNA from frozen OCT embedded tissues. First digest 10 to 30 thin sections in four milliliters of nucleic lysis buffer with protase K and 0.625%SDS in a 37 degrees Celsius water bath overnight. On the following day, add one milliliter of saturated five molar sodium chloride to the digestion mixture and shake vigorously for 15 seconds, centrifuge at 1, 100 times G for 15 minutes.

At room temperature, transfer the supinate to a new 15 milliliter centrifuge tube. Add two volumes of 100%ethanol add mix by inverting the tube six to eight times centrifuge at 5, 000 times G for 60 minutes at four degrees Celsius. To collect the precipitated DNA, wash the DNA pellet twice with 70%ethanol centrifuge at 5, 000 times G for 15 minutes each time.

To collect the pellet, dissolve the DNA pellet in 100 to 400 microliters of TE buffer at room temperature on a shaker overnight to extract DNA from thin sections of formin fixed paraffin embedded tissue. First de parize by incubating the paraffin sections in one milliliter of xylene twice at room temperature for 10 minutes each time. Collect the tissue sections by spinning at 13, 000 times G for five minutes at room temperature.

Next, remove residue elenes by incubating the sections in one milliliter of 100%Ethanol twice at room temperature for 10 minutes each time. Collect the tissue sections by spinning at 13, 000 times G for five minutes. At room temperature, air dry the sections at room temperature for 10 to 15 minutes.

Add a freshly prepared protease case solution to the samples in a final volume of 50 to 100 microliters and incubate overnight at 37 degrees Celsius on the following day. Heat the samples at 95 degrees Celsius for 10 minutes to inactivate the proteinase. K.The DNA yield from the extractions demonstrated in the previous segment is five to 200 micrograms depending on the size of the tissue.

The next step is to assess the quality of each DNA sample in a PCR tube mix. Point 25 microliters of TDNA polymerase with 45 microliters of master mix from a commercial ladder kit. Add five microliters of DNA into the PCR tube and mix well by pipetting up and down at least five times.

Amplify the DNA following the conditions detailed in the protocol text. Next, mix 20 microliters of the PCR reaction with four microliters of six x loading dye and load onto a 2%aros gel. When electrophoresis is complete stain the AGROS gel with Ethereum bromide and detect the PCR products.

With a gel imaging system, the samples that yield five PCR products at sizes of 100 200, 300, 400 and 600 base pairs will be used to generate VDJ amplicons only. The amplification of recombined I-G-H-V-D-J segment from framework region one or IGV HFR one will be demonstrated in this video. The amplification of recombined I-G-H-B-D-J segment from framework region two follows a similar procedure and is described in the protocol text in a PCR tube mix.

45 microliters of master mix from the tube labeled mix two of a commercial somatic IGH HYPERMUTATION assay for gel detection kit point 25 microliters of TAC DNA polymerase and five microliters of sample DNA amplify the DNA following the conditions described in the protocol text To amplify suboptimal D-N-A-P-C-R conditions can be adjusted by extending a kneeling time or increasing amplification cycle.Numbers. Resolve the entire PCR products by electrophoresis and a 2%AGROS gel stain. The AGROS gel with a 0.5 micrograms per milliliter Ethereum bromide solution and detect PCR products.

With a gel imaging system, a monoclonal amplicon is expected with the size range of 310 to 380 base pairs. Exci the gel portion containing the monoclonal amplicon. Subsequently purify the DNA from the excise to gel using a standard gel extraction kit according to the manufacturer's protocol.

To begin this procedure, transfer the purified V-D-J-P-C-R product into a PCR tube and add the Resus suspension buffer from the DNA sample preparation kit to bring up the volume to 60 microliters. Add 40 microliters of end repair mix from the DNA sample preparation kit and mix thoroughly incubate the reaction at 30 degrees Celsius for 30 minutes in a preheated thermocycler with a preheated lid at 100 degrees Celsius. Next, mix 136 microliters of magnetic beads and 24 microliters of PCR grade water in a 1.5 milliliter tube and transfer the entire end repair reaction into the tube and mix well with the bead solution.

Place the tubes on a magnetic stand for two minutes to allow the separation of the beads from the solution. Aspirate the sup natant and wash the beads twice with freshly prepared 80%ethanol. Aspirate the ethanol solution completely and allow the beads to air dry for 15 minutes at room temperature.

After 15 minutes, take the tubes off the magnetic stand and resuspend the beads in 17.5 microliters of Resus Suspension Buffer. Place the tubes back onto the magnetic stand for two minutes to separate the beads from the Resus Suspension Buffer. Transfer the Resus Suspension Buffer, which now contains the end repair product into a clean PCR tube.

The next step is to add the a tail to the end repair product. Add 12.5 microliters of the a tailing mix into the PCR tube containing the end repair product and mix thoroughly. Incubate the PCR tubes at 37 degrees Celsius for 30 minutes in a preheated thermocycler with a preheated lid at 100 degrees Celsius.

For adapter ligation, add 2.5 microliters of Resus suspension buffer, 2.5 microliters of ligation mix, and 2.5 microliters of DNA adapter index into the a tailing reaction. Incubate the reaction at 30 degrees Celsius for 30 minutes and a preheated thermocycler with a preheated lid at 100 degrees Celsius. After 30 minutes, add five microliters of stop ligation buffer into each reaction and mix thoroughly.

Add 42.5 microliters of well mixed magnetic beads to each reaction and wash with 80%ethanol as shown earlier. Add 50 microliters of Resus suspension buffer to elute the adapter ligated product. Clean the adapter ligated product for a second time by using 50 microliters of well mixed ampu XP beads and elute the product in 25 microliters of Resus Suspension Buffer into a clean PCR tube.

To amplify the VDJ Amplicon, add five microliters of PCR primer cocktail and 25 microliters of PCR master mix to the PCR tube containing the adapter ligated product and mix thoroughly perform amplification in a thermocycler with the conditions described in the protocol text. When the amplification is complete, clean up the reaction using 50 microliters of well mixed magnetic beads, elute the final product in 30 microliters of Resus Buffer. The VTJ Amplicon library is subsequently validated, pooled, and sequenced as described in the protocol text.

Because the complexity of the VVG sequencing library is low, we recommend to add in a 20 to 50%FI specing in approved sequencing samples to allow accurate based calling. These representative gel images show V-D-J-P-C-R Amplicon products from DNA extracted from lymphoma Patient samples. The quality of the V-D-J-P-C-R amplicon before and after library construction was assessed by a bioanalyzer.

The size shift from 334 base pairs to 459 base pairs indicates the addition of the adapter to trace the clonal evolution of a diffuse large B cell lymphoma relapse case. Phylogenetic analysis of the somatic hypermutation or SHM profiles of the major heavy chain VDJ rearrangements. Between the diagnosis and relapse samples was performed.

The late divergent clonal evolution mode was observed. New graphical methods were developed to visualize clonal evolution. Patterns shown are multidimensional scaling plots of a late divergent and an early divergent relapse model.

The radius of each circle indicates the count of sub clones corresponding to a particular SHM profile. Blue corresponds to the diagnosis sample, and red corresponds to the relapse sample. Likewise, undirected graphs show the distribution of sub clone counts in the late divergent case defers from the early divergent case.

The edges correspond to two SHM profiles having a string distance of one letter separation and the color scale bar indicates the proportion of sequences mapping to a particular SHM profile corresponding to the red diagnosis sample, or yellow relapse sample. While attempting this procedure, it's important to avoid cross contaminations between samples. After watching this video, you should have a good understanding of how to assess ality of lymphoma tumor samples.

This approach can also be used to trace mineral residual disease and probe immune surveillance response to cancers. Thanks for watching and good luck with your experiments.