The feature of VDJ rearrangment is that to maintain the specificity of recombination, V(D)J recombinase recognizes and binds to recombination signal sequences
(RSSs) flanking the variable (V), diversity (D), and joining (J) genes
segments. RSSs are composed of three elements: a heptamer of seven
conserved nucleotides, a spacer region of 12 or 23 basepairs in length,
and a nonamer of nine conserved nucleotides. While the majority of RSSs
vary in sequence, the consensus heptamer and nonamer sequences are
CACAGTG and ACAAAAACC, respectively; and although the sequence of the
spacer region is poorly conserved, the length is highly conserved.
The length of the spacer region corresponds to approximately one (12
basepairs) or two turns (23 basepairs) of the DNA helix. Following what
is known as the 12/23 Rule, gene segments to be recombined are usually
adjacent to RSSs of different spacer lengths (i.e., one has a "12RSS" and one has a "23RSS"). This is an important feature in the regulation of V(D)J recombination.[
A software, VDJSeq-Solver is a completely automated workflow for the in silico detection of the main clone V(D)J recombination sequence characterizing neoplastic tissues, using RNA-Seq paired-end reads.
B-cells and T-cells are different from other cells in the fact that their genomes bear a genomic birthmark of diversity. They can expand under specific conditions and form monoclonal populations bearing identically rearranged gene segments. These clonal populations are usually under tight control mechanisms. However, under special occasions they might expand to an extent which causes a
disease, such as in autoimmune disorders, leukemias and lymphomas: It is nowadays note that knowing the precise sequence of rearranged BCR genes provides lots of useful information both from an investigative and from a clinical point of view.
Among Next Generation Sequencing (NGS) approaches, RNA sequencing (RNA-Seq) can theoretically identify sequences and quantitate every RNA fragment present in a sample. In theory, it should be an ideal method to identify clonal lymphocyte populations (also in the context of a polyclonal background), since the number of reads mapping to the genes rearranged in the neoplastic clone should be much larger than those mapping to other BCR or TCR genes. Moreover, precise information concerning the rearranged BCR or TCR genes of the dominant clone should be available at the same time, therefore
promising to be the most complete clonality test.
Using a set of paired-end RNA-Seq reads, derived from the sequencing of lymphoma mRNA samples,
VDJSeq-Solver firstly identifies the main clone characterizing the tissue of interest by detecting the most amplificated V(D)J rearrangement of the sample under study. Then considering how reads are mapped on the V, D and J alleles involved in the selected rearrangement, the specific sequence of the clone is reconstructed.
A published patent data dealing with detection of VDJ rearrangment of which the use of sequence specific enrichment and specific restriction
endonuclease enzymes or other DNA-shearing approaches to provide high
resolution and high throughput interrogation of antigen receptor