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)
Figure 1: Schematic representation of VDJ recombination and primer design for amplification of the VDJ region.
See figure in Figures section. Figure 1: Schematic representation of VDJ recombination and primer design for amplification of the VDJ region. (A) The heavy chain immunoglobulin locus prior and (B) 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:
Table 2: Components for 1st PCR reaction
See figure in Figures section. 8| Start PCR reaction using the following cycler program:
Table 3: Cycler program for 1st PCR reaction
See figure in Figures section. Purification of the 1st PCR reaction
9| Preparations before start of the purification
(i) The optimal working temperature for the AMPure XP beads is on room temperature. Keep in mind to take them out of the fridge in time. 20 µl AMPure XP beads per sample are needed. Vortex AMPure XP beads for 30 seconds to generate a homogenous solution
(ii) Bring 10 mM Tris-HCl (pH 8.5) solution to room temperature. 52.5 µl is needed per reaction.
(iii) Prepare 80% ethanol solution freshly on room temperature. 400 µl is needed per sample.
10| Add 20 µl AMPure XP beads to each PCR reaction and mix well by pipetting up and down until beads are resuspended
11| Incubate the mixture for 5 min at room temperature
12| Place the samples on a magnetic stand (e.g. DynaMag-96 Side Magnet) and wait until the beads form a pellet and the supernatant is clear
13| Remove the supernatant without destroying the bead pellet
14| Keep samples on the magnetic stand while washing twice with 200 µl 80% ethanol solution. Remove ethanol after 30 seconds incubation without destroying the bead pellet.
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:
Table 4: Components for 2nd PCR reaction
See figure in Figures section. 23| Start PCR reaction using the following cycler program:
Table 5: Cycler program for 2nd PCR reaction
See figure in Figures section. 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.
Determination of DNA quantity and quality
25| 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 450 bp. Representative TapeStation electropherograms of a good and a critical run are shown in Figure 2.
Troubleshooting!
Figure 2: Representative TapeStation electropherograms
See figure in Figures section. Figure 2: Representative TapeStation electropherogram of a good (top) and a critical (bottom) run of the 2nd PCR reaction and AMPure bead purification.
26| Determine the peak molarity [nmol/l] of the PCR product by integrating under the peak. Calculate the volume inserted for the indexing PCR (Step 27) as follows:
Table 6: Volume for the indexing PCR dependent on peak molarity
See figure in Figures section. 3rd PCR amplification for indexing of samples for Illumina Sequencing
27| Set up indexing PCR reaction in 0.2 ml PCR tubes as follows:
Table 7: Components for indexing PCR reaction
See figure in Figures section. CRITICAL STEP: All samples sequenced on one flow cell must have unique Index combinations
28| Start PCR reaction using the following cycler program:
Table 8: Cycler program for indexing PCR reaction
See figure in Figures section. 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
Figure 3: Representative TapeStation electropherograms
See figure in Figures section. Figure 3: Representative TapeStation electropherogram of a good (top) and a critical (bottom) run of the indexing PCR reaction and AMPure bead purification of 1:20 diluted samples.
31| Determine the DNA concentration [ng/µl] of the purified PCR product (Step 29 (ii)) of the indexing PCR using the Qubit dsDNA HS Assay Kit according to the manufacturer’s instructions.
32| Calculate the DNA concentration in nM as follows:
Equation 1: Calculation of DNA concentration in nM
See figure in Figures section. Pooling and denaturation of the libraries
33| Dilute each sample to a final concentration of 4 nM in 10 mM Tris-HCl (pH 8.5) in DNA LoBind Tubes.
34| Pool equimolar amounts, e.g. 5 µl per sample in a DNA LoBind Tube. Depending on the desired coverage, up to 3 x 96 samples can be pooled for one run on a MiSeqDx system using MiSeq Reagent Kit v3 (600-cycle) in a 300 bp paired-end run. In this setup with 96 multiplexed samples, a read depth of ~ 250.000 reads per sample is achieved.
35| Determine DNA concentration [ng/µl] of the library pool (Step 34) using the Qubit dsDNA HS Assay Kit according to the manufacturer’s instructions.
CRITICAL STEP: The concentration of the pool should be 4 nM.
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.
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:
Table 9: MiSeqDx system run parameter
See figure in Figures section. 42| Sequence with the MiSeq Reagent Kit v3 (600-cycle).
Analysis of the B cell receptor repertoire
42| For bioinformatic analysis the MiXCR software v2.1.8 and the reference library repseqio.v1.4 was used. Change the species parameter setting to Mus musculus; the default setting is Homo sapiens.