Exome sequencing of the TCL1 mouse model for CLL reveals genetic heterogeneity and dynamics during disease development

The TCL1 mouse model is widely used to study pathophysiology, clonal evolution, and drug sensitivity or resistance of chronic lymphocytic leukemia (CLL). By performing whole exome sequencing, we present the genetic landscape of primary tumors from TCL1 mice and of TCL1 tumors serially transplanted into wild-type recipients to mimic clonal evolution. We show that similar to CLL patients, mutations in mice are frequently subclonal and heterogenous among different primary TCL1 mice. We further describe that this molecular heterogeneity mirrors heterogenous disease characteristics such as organ infiltration or CLL dependent T cell skewing. Similar to human CLL, we further observed the occurrence of novel mutations and structural variations during clonal evolution and found plasticity in the expansion of B cell receptor specific subclones. Thus, our results uncover that the genetic complexity, pathway dependence and clonal dynamics in mouse CLL are in relevant agreement to human CLL, and they are important to consider in future research using the TCL1 mouse for studying CLL.


Introduction
In the context of cancer, analysis of the BCR repertoire in mice becomes increasingly important to define clones and subclones within a tumor population. There are several approaches for this analysis, e.g. by spectratyping, but high-throughput sequencing is the state-of-the-art nowadays. So far, there are no manufacturers kits for mouse BCR repertoire analysis by NGS available. Therefore, we developed an in-lab protocol with self-designed degenerated primers for amplification of the rearranged VDJ locus of the immunoglobulin heavy chain using genomic DNA as input material. With this protocol, up to 3×96 mouse samples can be multiplexed and subjected to Illumina sequencing in a fast and cost-saving way.

REAGENT SETUP 80% ethanol
Prepare an 80% ethanol solution (vol/vol) with nuclease-free water freshly for each purification reaction by mixing 1 part ethanol absolute for analysis and 4 parts nuclease-free water.

Procedure
Primer design for amplification of the VDJ region of the B-cell receptor 1| Nucleotide sequences of the mouse IGHV and the IGHJ region can be obtained from the IMTG genome database (http://www.imgt.org/vquest/refseqh.html#VQUEST) 2| Perform a sequences alignment of each gene segment (IGHV and IGHJ) and create a frequency plot with WebLogo (https://weblogo.berkeley.edu/logo.cgi) 3| Primer sequences were obtained based on frequencies of single bases within the aligned sequence using the UIB code for degenerated primers (see Figure 1) (i) For the IGHV gene segment the sequence alignment of the framework region 1 (FR1) clustered into 2 groups, hence 2 different degenerated forward primers were generated (see Table 1, RG1269 and RG1270) (ii) For the IGHJ gene segment 1 degenerated reverse primer was generated (see Table 1, RG1271)  after VDJ recombination, including detailed representation of the rearranged VDJ segments with framework region 1-3 (FR 1-3) and complementarity-determining regions 1-2 (CDR1-2) on the V segment, CDR3 on the rearranged VDJ segments and FR4 on the J segment. (C) Frequency plots of the primer binding sites created in WebLogo and the corresponding primer sequences in bold.

Quantification of genomic DNA (gDNA)
4| Determine gDNA concentration of CD5+/CD19+ sorted CLL cells using the Qubit dsDNA HS Assay Kit according to the manufacturer's instructions. 5| 30 ng of gDNA is used as a template for PCR amplification in Step 7.
Amplification of the VDJ region of the B-cell receptor by PCR 6| Prepare primer mix for 1st PCR reaction (Phusion POL) (i) Dilute primer stocks (100 µM) of RG1269, RG1270 and RG1271 to a final concentration of 10 µM using nuclease-free water, e.g. mix 5 µl of each 100 µM primer stock with 45 µl of nuclease-free water.
(ii) Mix equal volumes of each primer to generate a primer mix with 10 µM of each primer 7| Set up 1st PCR reaction in 0.2 ml PCR tubes as follows:  15| Make sure to remove all remaining ethanol after each wash step  CRITICAL STEP: Removal of as much ethanol as possible is critical here, to ensure the removal of residual primers of the 1st PCR reaction that could interfere with the 2nd PCR reaction. In addition, it is important to not destroy the bead pellet or lose beads by washing them out which leads to lower yields of the PCR product after elution. 16| Incubate on room temperature for 10 min until residual ethanol is evaporated, but do not over dry the bead pellet. The pellet should appear dry but not broken or cracked. 17| Take samples from magnetic stand and resuspend the dry bead pellet in 52.5 µl 10 mM Tris-HCl (pH 8.5) until the solution is homogenous 18| Incubate the mixture for 5 min at room temperature 19| Place the samples back on the magnetic stand and wait until the beads form a pellet and the supernatant is clear 20| Transfer 50 µl of the supernatant into a new PCR tube without destroying the bead pellet Troubleshooting! OPTIONAL: Analyze samples on a D1000 ScreenTape in a TapeStation system to monitor successful amplification and removal of residual primers. The PCR product should have a length of around 370 bp.
 PAUSE POINT: Reactions can be stored at -15° to -25°C for up to one week.

2nd PCR amplification for tagging of the VDJ region of the B-cell receptor
21| Prepare primer mix for 2nd PCR reaction (Phusion POL) (i) Dilute primer stocks (100 µM) of RG1272, RG1273 and RG1274 together in one tube to a final concentration of 1 µM using nuclease-free water, e.g. pipette 10 µl of each 100 µM primer stock together with 970 µl of nuclease-free water. 22| Set up 2nd PCR reaction in 0.2 ml PCR tubes as follows:  Purification of the 2nd PCR reaction 24| Repeat step 9 to 20 to purify the PCR product of the 2nd PCR reaction  PAUSE POINT: Reactions can be stored at -15° to -25°C for up to one week.    Purification of the indexing PCR reaction 29| Repeat step 9 to 20 to purify the PCR product of the indexing PCR reaction with minor modifications (i) Use 27.5 µl of 10 mM Tris-HCl (pH 8.5) for resuspension of the beads (Step 17) (ii) Transfer 25 µl of the supernatant into a new DNA LoBind Tube (1.5 ml) without destroying the bead pellet (Step 20)  PAUSE POINT: Reactions can be stored at -15° to -25°C for up to one week Quantification of indexed DNA 30| Analyze 1:10 to 1:50 diluted samples (10 mM Tris-HCl, pH 8.5) on a D1000 ScreenTape in a TapeStation system to monitor successful amplification and removal of residual primers. The PCR product should have a length of around 520 bp. Check whether the main peak is above 90 % of the integrated area and no impurity occurs. Representative TapeStation electropherograms of a good and a critical run are shown in Figure 3. Troubleshooting! CRITICAL STEP: Do not dilute the whole sample, only some µl for analysis in a TapeStation system 36| For denaturation of the library pool combine 5 µl of the 4 nM library pool with 5 µl of 0.2 N NaOH (freshly prepared) in a DNA LoBind Tube, mix by vortexing, spin down and incubate at room temperature for 5 min. 37| Add 990 µl pre-chilled HT1 buffer to obtain a 20 pM denatured library, keep the library on ice. 38| Dilute the denatured library to 8 pM by combining 240 µl of the 20 pM denatured library (Step 37) with 360 µl pre-chilled HT1 buffer. 39| Keep the diluted and denatured library on ice until sequencing.

Determination of DNA quantity and quality
Sequencing on a MiSeqDx system 40| Prepare the MiSeqDx system for a sequencing run according to the manufacturer's instructions. 41| Use the Illumina Experiment Manager Software in FASTQ only Run Settings to generate the sample sheet using the following parameters:

Timing
Total time: ~ 5 days Step 1-3 Primer design for amplification of the VDJ region of the B-cell receptor -3 h Step 4-5: Quantification of genomic DNA (gDNA) -1h Step 6-8: Amplification of the VDJ region of the B-cell receptor by PCR -1 h Step 9-20: Purification of the 1st PCR reaction -1.5 h 21-23: 2nd PCR amplification for tagging of the VDJ region of the B-cell receptor -1 h 24: Purification of the 2nd PCR reaction -1.5 h 25-26: Determination of DNA quantity and quality -1 h 27-28: 3rd PCR amplification for indexing of samples for Illumina Sequencing -1 h 29: Purification of the indexing PCR reaction -1.5 h 30-32: Quantification of indexed DNA -1.5 h 33-39: Pooling and denaturation of the libraries -1.5 h 40-41: Sequencing on a MiSeqDx system -2 d 42: Analysis of the B cell receptor repertoire -1 d The timing information is based on the preparation of 24 samples, for more samples consider more time.

Anticipated Results
Fast and precise analysis of the amino acid sequence of the BCR CDR3 region in mice by highthroughput sequencing on an Illumina MiSeqDx system.