NGS-Based B-Cell Receptor Sequencing Protocol

Amplification of VH-Cg, VH-Ca, or VH-Cμ from cDNA

1. Prepare PCR master mix consisting of 28.3 μl water, 5 μl 10× Buffer Gold, 3 μl MgCl2, 0.5 μl dNTPs, 1 μl BSA, and 0.2 μl Taq Gold.
2. Transfer PCR master mix into PCR reaction tubes (38 μl into each well).
3. Deposit 1 μl of each primer into the corresponding well.
4. Add 5 μl cDNA to the corresponding well, and carefully add the lid of the PCR tubes.
5. Run PCR at 95 °C for 7 min; 25–35 cycles at 94 °C for 30 s, 57 °C for 30 s, 72 °C 1 min; 72 °C for 10 min.
6. Load 50 μl PCR product with 10 μl loading dye onto a 1% agarose gel in TBE buffer containing ethidium bromide and run for 1 h at 180 V.
7. Visualize DNA band under ultraviolet (UV) light, and cut the PCR band of approximately 500 bp from gel using a scalpel.
8. Purify the PCR product from gel using the gel extraction kit. Follow the instructions in the manual and eluate with 20 μl elution buffer.
9. Continue with Subheading 3.3, Nested PCR.

Amplification of VH-JH from DNA

1. Prepare PCR master mix consisting of 31.3 μl water, 5 μl 10× Buffer Gold, 3 μl MgCl2, 0.5 μl dNTPs, 1 μl BSA, and 0.2 μl Taq Gold.
2. Transfer PCR master mix into PCR reaction tubes (41 μl into each well).
3. Deposit 1 μl of each primer (6 5′ primers and 1 Cg or Ca primer per well) into the corresponding well.
4. Add 2 μl 50 ng/μl DNA to the corresponding well, and carefully add the lid of the PCR tubes.
5. Run PCR at 95 °C for 7 min; 25–35 cycles at 94 °C for 30 s, 57 °C for 30 s, 72 °C 1 min; 72 °C for 10 min.
6. Load 50 μl PCR product with 10 μl loading dye onto a 1% agarose gel in TBE buffer containing ethidium bromide and run for 1 h at 180 V.
7. Visualize DNA band under ultraviolet (UV) light (see Note 7), and cut the PCR band of approximately 500 bp from gel using a scalpel.
8. Purify the PCR product from gel using the gel extraction kit. Follow the instructions in the manual and eluate with 20 μl elution buffer.

Nested PCR and Pooling

1. Add 12.5 μl KAPA HiFi Hotstart Ready mix, 2 μl TruSeq Custom Amplicon Index forward primer, 2 μl TruSeq Custom Amplicon reverse primer, and 8.5 μl purified PCR product to a PCR reaction tube.
2. Run PCR at 95 °C for 5 min; 10 cycli at 98 °C for 20 s, 66 °C for 30 s, 72 °C 30 s; 72 °C for 1 min.
3. Measure the concentration of the PCR products.
4. Mix the PCR product at an equimolar concentration of 50 mM.
5. Purify the pool of PCR products.
6. The PCR pool can be sequenced using the Illumina platform.

Merging, Trimming, and Alignment of Reads Using Galaxy

1. Sequencing with the Illumina platform results R1 and R2 reads that need to be merged before they can be aligned to a reference database.
2. After the reads are merged, the Illumina Rd1 and Rd2 primer adapters have to be removed from the reads as well as the forward primers.
3. Before the reads can be aligned using IMGT/HighV-Quest, the FASTQ files have to be adapted to the FASTA file format. This can be done with the FASTQ to FASTA converter.
4. For alignment and annotation of the BCR rearrangements, the international ImMunoGeneTics system IMGT/HighV-Quest can be used.

Data Analysis Using the Immune Repertoire Pipeline in Antigen Receptor Galaxy

1. Open ARGalaxy.
2. Upload the compressed .txz files using: get data → upload file. The file will appear on the right site of your screen under "History."
3. Select under "Tools" on the left site of the screen "ARGalaxy," and click on the "Immune repertoire pipeline."
4. Select the .txz file you would like to analyze.
5. Enter a name in the "ID" field.
6. Select the definition of the clonotype.
7. Select the order in which the V, D, and J genes have to appear in the graphs. The default setting is on alphabetical order and not in the order they appear on the IGH locus.
8. Select "IGH " at the "Locus" field.
9. Choose if you want to visualize the unproductive rearrangements in the graphs.
10. Select if you want to identify overlapping sequences between different replicates within one donor.
11. Press execute. A new item will be displayed in your history and turn green when the tool is ready with processing the data.
12. Click on the "eye" symbol to open the table that shows an overview of the rearrangements, including the percentage of productive, productive unique, unproductive, and unproductive unique.
13. Press on "Click here for the results" to open the page with the different analysis tabs.
14. The tab "Gene frequencies" shows the percentage of V, D, an J gene usage. The frequency of the different V, D, and J genes vary slightly between individuals and also between different primer sets that are used to amplify the BCR rearrangements.
15. The tab "CDR3 characteristics" contains plots that show the distribution of the CDR3 length and the frequency of the different amino acids used in the CDR3. The median CDR3 length is longer in naïve B cells compared to memory B cells, which is likely caused by selection against long CDR3 lengths, because they are more likely to be autoreactive.
16. In the tab "Heatmaps," the frequency of the different combinations of V-J, V-D, and D-J genes are visualized in heatmaps.
17. In the tab "Compare heatmaps," the heatmaps between different donors can be compared.
18. In the tab "Circos," the frequency of the different combination of V-J, V-D, and D-J genes are visualized using circus plots.
19. When the option is chosen to determine the number of sequences that share the same clonal type between replicates or to determine the clonality of the donor, the tab "Shared Clonal Types" or "Clonality" is shown. These tabs include a table with information about the number of BCR rearrangement that is present in multiple replicates of the same donor. When three replicates are present and the option "determine the clonality of the donor" is chosen, the clonality score based on the publication by Boyd et al. is given. In patients with IEI, the diversity of the repertoire is often reduced.
20. The tab "Junction analysis" contains a table with the median or mean number of deletions, palindromic (P) nucleotides, or non-templated (N) nucleotides in the productive and unproductive rearrangements. Genetic defects in the non-homologous end joining (NHEJ) pathway have been shown to affect the number of deletions, N-nucleotides and P-nucleotides.
21. In the "Download" tab, all data used to create the tables and graphs can be downloaded. 

Reference:

1. Schouwenburg P A, Burg M, IJspeert H. NGS-Based B-Cell Receptor Repertoire Analysis Repertoire analyses in the Context of Inborn Errors of Immunity[M]//Immunogenetics. Humana, New York, NY, 2022: 169-190.