<scp>ARTISAN PCR</scp>: rapid identification of full‐length immunoglobulin rearrangements without primer binding bias (2016)

B cells recognize specific antigens by their membrane-bound B-cell receptor (BCR). Functional BCR genes are assembled in pre-B cells by recombination of the variable (V), diversity (D) and joining (J) genes [V(D)J recombination]. When B cells participate in germinal centre reactions, non-templated point mutations are introduced into BCR genes by somatic hypermutation (SHM) (Rajewsky, 1996). V(D)J recombination and SHM create virtually unlimited BCR repertoires. Many research applications require identification of full-length BCR sequences expressed in biological samples. BCR are commonly identified by multiplex polymerase chain reaction (PCR) with upstream primers that anneal to conserved stretches in variable or leader gene segments. BIOMED-2 is the current European standard protocol to assess BCR clonality as diagnostic criterion for lymphoma (van Dongen et al, 2003). However, a single point mutation in a primer binding site can obscure a dominant clonal BCR (Wu et al, 2009), whereas reliable identification of functional BCR genes is essential to avoid erroneous conclusions about the BCR (Koning et al, 2014). Therefore, several investigators have emphasized the need for an unbiased method of BCR amplification (Warren et al, 2013; Georgiou et al, 2014; Koning et al, 2014). We here describe ARTISAN PCR (Anchoring Reverse Transcription of Immunoglobulin Sequences and Amplification by Nested PCR) as a truly unbiased method with excellent applicability. Polyadenylated mRNA (Dynabeads mRNA DIRECT Kit, Invitrogen, Grand Island, NY, USA) was directly isolated from thawed suspensions of cryopreserved cells, or from 10 μm sections of fresh frozen material that was homogenized immediately upon addition of lysis buffer. Isolation of B cells by magnetic beads prior to mRNA isolation was found to be dispensable. Reverse primers (Table SI) were designed to anneal to a consensus sequence from all functional alleles of μ, γ, α, κ, and λ Ig constant regions included in the ImMunoGeneTics (IMGT) database (Lefranc et al, 1999). Messenger RNA was mixed with reverse transcription primers and the SA.rt anchor oligonucleotide (Table SII). Synthesis of cDNA was performed with a reverse transcriptase that adds a non-templated 3′ polycytosine terminus (Zhu et al, 2001). Annealing of the SA.rt oligonucleotide to this polycytosine stretch creates a template for extension of the cDNA with the complementary SA.rt sequence in the same reaction. Subsequent PCR amplification of anchored cDNA was performed with anchor-specific primer SA.pcr in combination with a nested primer that anneals to one of the constant regions of interest (Cμ.pcr, Cγ.pcr, Cα.pcr, Cκ.pcr, or Cλ.pcr; Table SI and Table SII). The efficacy of ARTISAN PCR was compared with a BIOMED-2-based multiplex PCR kit (IGH FR1, IGK tube A and IGL Clonality Assays, Invivoscribe, San Diego, CA, USA). For massive parallel sequencing, column-purified ARTISAN amplicons were barcoded by PCR (Table SII). Samples were sequenced on the PacBio RS system (Pacific Biosciences, Menlo Park, CA, USA). After filtering for a minimum of eight passes (SMRT Portal software, Pacific Biosciences) and correction of indels in polyhomologous stretches (Koren et al, 2012), sequence reads were analysed with Geneious 6.1.6 software and IMGT HighV-QUEST tools (Alamyar et al, 2012). PacBio sequencing of ARTISAN PCR amplicons identified full-length functional V(D)J sequences in all 39 Epstein–Barr virus-transformed lymphoblastoid cell lines (EBV LCL) tested (Table SIII). 103 cells sufficed as starting material. Each PacBio SMRT cell yielded >25 × 103 reads with at least eight passes. Of 52 VDJ sequences, 50 were functional and 2 non-productive. Thirty-two EBV LCL were monoclonal and were six biclonal; one EBV LCL harboured six different B-cell clones. The sequence error rate as calculated for clonal EBV LCL sequences was 0·126 × 10−3 per bp without significant differences between μ, γ, α, κ, and λ amplicons. The multiplex protocol failed to amplify seven VDJ, one VJ-κ and nine VJ-λ, indicating success rates of 87%, 95%, and 78%, respectively, compared to ARTISAN PCR. Complete BCR heterodimers were obtained by the multiplex protocol for only 37 of the 50 functional EBV LCL clones identified by ARTISAN PCR (74%). Dilution experiments with B-cell lines demonstrated proportional BCR sequence representation according to cellular input (Fig 1A). Anchoring Reverse Transcription of Immunoglobulin Sequences and Amplification by Nested PCR identified complete functional BCR sequences from cryopreserved primary cells in all 24 cases of various B cell (pre)malignancies (3 samples each from Burkitt lymphoma, mantle cell lymphoma, marginal zone lymphoma, follicular lymphoma, multiple myeloma, Waldenström macroglobulinaemia, chronic lymphocytic leukaemia and monoclonal B-cell lymphocytosis; Table SIV). Multiplex RT-PCR failed to identify four VDJ and eight VJ sequences, and identified the complete BCR heterodimer in only 13 cases (54%). Finally, ARTISAN PCR was applied to 17 fresh frozen excisional biopsies archived between 1988 and 1991 and labelled with diagnoses consistent with diffuse large B-cell lymphoma. For 15 biopsies, dominant clonal sequences were identified for the heavy as well as the light BCR chain. In two cases, neither the heavy nor the light chain amplicons included clonal sequences. Pathological revision confirmed lymphoma infiltration in the 15 samples with clonal BCR sequences but demonstrated lack of lymphoma infiltration in both non-clonal biopsies. The multiplex approach failed to amplify three VDJ and three VJ clonal rearrangements, yielding complete BCR heterodimers for 10/15 (67%) cases only (Fig 1B, Table SV). Across all experiments, functional clonal BCR sequences found by multiplex reverse transcription (RT)-PCR were also identified by ARTISAN PCR. Conversely, ARTISAN PCR identified specific causes for each multiplex RT-PCR failure: mismatches in primer binding sites (n = 25), lack of a primer for certain IGLV alleles (n = 9), and one truncation of the IGHV3-33*01 F gene (Fig 2A). Failure of multiplex RT-PCR correlated with SHM mutation frequency. When only IGV alleles for which a primer was designed in BIOMED-2 were considered, multiplex RT-PCR failed for 36% of sequences with <90% identity to germline (Fig 2B). When considering all IGV alleles, the failure rate rose to 42% of sequences with high SHM load (Fig 2C). Anchoring Reverse Transcription of Immunoglobulin Sequences and Amplification by Nested PCR, combined with a massive parallel sequencing technology that covers entire V(D)J sequences in a single read, e.g. the PacBio platform, permits unbiased comprehensive analyses of BCR repertoires in healthy, reactive, or neoplastic conditions. ARTISAN PCR has low error and apparently complete success rates but requires intact RNA as starting material for the protocol described here. In routine diagnostics performed on formalin-fixed tissue, amplification of DNA with BIOMED-2 primers has the advantage of feasibility but is limited by a substantial failure rate for hypermutated BCR sequences. The technical support of the Leiden Genome Technology Centre (LGTC) for processing of PacBio samples and advice on methodological issues is gratefully acknowledged. This research was funded by grants from the Professor Steenhuis Fonds, Leiden, SASC start-up grant No 7, and FONDECYT No 11140542 (M.A.N.). H.V. and M.A.N. designed the study; P.M.K. and C.A.M.v.B. collected and archived biopsy material; M.K. and V.B. performed the experiments; M.K., S.K. and S.A.J.v.d.Z. analysed the data; H.P.J.B. and M.G. advised on methodology; M.K., H.V. and M.A.N. wrote the manuscript. The authors have no conflicts of interest to report. Table SI: Oligonucleotide primers. Table SII: ARTISAN PCR and Barcoding Reactions. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.