B-Cell Receptor Gene Rearrangement by Next Generation Sequencing, Blood

Immunoglobulin (Ig) genes undergo somatic gene rearrangement resulting in a diversity of antigen binding capability among B-cells. The IG is a tetrameric protein consisting of two highly variable heavy chains (IgH) and light chains (IgL and/or IgK). IG gene rearrangements occur early in B-cell ontogeny and are unique to each B-cell. These rearrangements can therefore be used as a marker of a common origin, or clonality, among a B-cell population and can be useful in the evaluation of hematopoietic disorders.

The immunoglobulin (Ig) heavy-chain locus (IGH) is on chromosome 14 (14q32.3) and includes 123-129 Vh (variable) gene segments group into seven families followed by 27 Dh (diversity) genes, 9 Jh (joining) genes, and 11 Ch (constant) regions spreading over 1250 kilobases (kb). The Vh gene segments are consist of three conserved frameworks (FR1,2, and 3).

The immunoglobulin (Ig) kappa light chain(IGK) gene locus on chromosome 2 (2p11.2) and includes 76 Vk (variable) gene segments group into seven families and five Jk gene segment spread over 1820 kilobases. In addition, the kappa deleting element (Kde) can rearrange with Vk or Jk-Ck Intron (INTR) and produce unsuccessful products remained in the B-cell immune repertoire.

Although standardized PCR/capillary electrophoresis (CE)-based assays (described by the BIOMED-2 concerted action study) have been widely adopted as the current gold standard in clonality testing, next-generation sequencing methods (NGS) have emerged with additional benefits. With the NGS approach, the exact DNA sequence specific for each clonal gene rearrangement can be identified, which offers a unique opportunity to track identified clones in subsequent samples. Clonal populations that may differ in sequence but are the same size can also be readily differentiated with NGS. In addition, the CE-based methods are not quantitative in nature, which often imposes difficulty in the interpretation of results.

The Invivoscribe LymphoTrack assays are targeted next generation sequencing (NGS)-based methods that enable quantitative identification of B-cell populations using a single multiplex master mix of primers targeting frameworks (FR1,2, and 3) of the conserved V(D)J regions of the immunoglobulin heavy chain gene locus (IGH) and immunoglobulin kappa light chain (IGK) gene locus. In addition, the kappa deleting element (Kde) and Jk-Ck Intron (INTR) can be detected by the IGK assay. Primers included in the master mixes are designed with Illumina adapters and up to 24 different indices, allowing for one-step PCR and pooling of amplicons from several different samples and targets. Each reaction mix contains consensus primers that are tagged with one of the 24 unique Illumina barcodes and Illumina® P5 and P7 flow cell adapter oligo sequences. This design allows for multiplexing of up to 24 samples and of multiple targets of interest. The samples can then be bioinformatically identified based on the sample-specific barcodes, and reads belonging to the targets of interest can be differentiated based on alignment to the reference sequence.

After PCR, amplicons are purified using a solid-phase reversible immobilization (SPRI) paramagnetic bead technology. DNA fragments greater than 100 bp are selectively bound to the paramagnetic beads while excess primers, unincorporated nucleotides, salts, and enzymes are removed by successive ethanol washes. The purified PCR products are eluted and quantified by KAPA Library Quantification Kits. Equimolar quantities of each library are pooled and quantified again by KAPA Library Quantification Kits. The primers in KAPA kit target Illumina P5 and P7 flow cell adapter oligo sequences from the amplicons. Only fragments that include an inserted fragment between the adapters will be quantitated by this method. The concentration of pooled library is determined by plotting its Ct score against a standard curve generated by pre-diluted DNA standards. The library is then loaded onto a flow cell on the Illumina MiSeq platform, where it is subjected to bridge amplification and sequencing by synthesis. The output data can then be analyzed with the LymphoTrack software, where the reads are sorted and identified by their unique sequence information. Clonality can then be determined based on the prevalence of identified sequences.

The analytical sensitivity by our validation studies is at least 5% clonal detection in a background of polyclonal distribution. A minimum of 50,000 reads per sample must be obtained from each sample as a QC requirement.