IMGT Education

Sequence analysis

Compare the V-J junction sequence with the extremities of the corresponding V and J "germline" genes (see IMGT Repertoire > Alignments of alleles).
It is the case of V and J genes of IGK, IGL, TRA and TRG loci.

Example: the rearranged sequence with accession number AB006843

When we read the IMGT annotations (in IMGT/LIGM-DB), we note that this rearranged sequence of a kappa light chain comes from the genes and the alleles rearrangement: IGKV2-28*01 [or IGKV2D-28*01 (identical score)] and IGKJ2*01.

If, in IMGT Repertoire > Alignments of alleles, we look at the V and J germline genes from which the sequence derives, we observe:
- somatic mutations (some nucleotides differ from the germline sequence)
- deletions at the 3' end of V gene and at the 5' end of J gene: 3 nucleotides deletion TCC at the 3' end of the IGKV2-28*01 allele and 2 TG nucleotides deletion at the 5' end of the IGKJ2*01 allele.
- nucleotide addition of nucleotides not coded into the germline sequence: one C nucleotide added.
Compare the V-D-J junction sequence with the extremities of the corresponding V, D and J "germline" genes.
It is the case of the V, D and J genes of IGH, TRB and TRD loci.

Example: the rearranged sequence with accession number AF052379.

When we read IMGT annotations (in IMGT/LIGM-DB), we note that this rearranged sequence of an heavy ../_FRchain comes from the genes and alleles rearrangement: IGHV5-51*03, IGHD1-7*01 and IGHJ4*02.

If, in IMGT Repertoire > Alignments of alleles, we look at the V, D and J germline genes from which the sequence derives, we observe:
- somatic mutations (some nucleotides differ from germline sequence)
- deletions at the 3' end of V gene, on both sides of D gene and at the 5' end of J gene: 2 nucleotides deletion at the 5' end of the IGHD1-7*01 allele, 4 nucleotides deletion at the 5' end of IGHJ4*02 allele.
  In other sequences, we could also observe deletions at the 3" end of the V gene.
- nucleotide addition of nucleotides not coded in the germline sequence, N-diversity regions, N1 and N2: insertions of 2 nucleotides CT (N1) between V and D, and insertion of 5 nucleotides TCTCG (N2) between D and J.
Is it easy to identify a D gene? Why?

No, it is not easy to identify and locate a D gene in a V-D-J junction. Indeed, it is a short gene (only a few tens nucleotides) which may have somatic mutations, deletions and nucleotide insertions (N-diversity) at the 5' and 3' ends.

Search in IMGT/LIGM-DB

Search of sequences
Request example:
- human
- productive
- Ig
- anti-TPO
Results example:

268 sequences (01/07/2003)
Request on a label
Request example: L-REGION

Results example:

22 sequences (01/07/2003)

Analysis of a flat file

ID:	IDentification line
AC:	ACcession number line
DT:	DaTe line
OS:	Organism Species
OC:	Organism Classification
DE:	DEscription line
KW:	KeyWord lines
FT:	Feature Table lines
R -:	RN: Reference Number, RC: Reference Comment, RP: Reference Positions, RX: Reference Cross-reference, RA: Reference Author(s), RT: Reference Title, RL: Reference Location
SQ:	SeQuence header

Using IMGT/V-QUEST

Note on a piece of paper the V, D and J gene and allele names:
Example:
IGHV1-69*06
IGHD3-10*01
IGHJ6*01
Example: sequence with accession number M11954
- TRBV20-1*03
- TRBD2*02
- TRBJ2-3*01
Locate the nucleotide sequence of the junction [from the cystein (c) codon at position 104 to the tryptophan (W) or phenylalanine (F) codons at position 118]. F or W is identified by the characteristic pattern F/W-G-X-G of the J gene.
Keep the IMGT/V-QUEST window and open a second one (File > New Window).
In IMGT/LIGM-DB, the option "9 IMGT/V-QUEST analysis" allows to obtain the translation of JUNCTION as follows:
```
Translation of the JUNCTION
             104                                             118
              C   S   A   K   L   V   G   T   D   T   Q   Y   F   G   P   G
M11954       TGC AGT GCT AAA CTA GTA GGT ACA GAT ACG CAG TAT TTT GGC CCA GGC
```

Using IMGT/JunctionAnalysis

Consult IMGT/JunctionAnalysis Documentation to see how to present your request into the window.
An example is described.
Copy in the window:
- on the first line, the required information (note: it is not necessary to indicate the D-GENE when it exists)
  
  >M11954,TRBV20-1*03,TRBJ2-3*01
- on the next line, the nucleotide sequence of the junction (obtained from IMGT/V-QUEST) (note: with or without spaces)
```
TGC AGT GCT AAA CTA GTA GGT ACA GAT ACG CAG TAT TTT
```
Results analysis:

Result example:
- number of deleted nucleotides in 3' of V, in 5' and 3' of D and in 5' of J
  - no deleted nucleotides at the 3' end of V-REGION
  - 3 deleted nucleotides at the 5' end of D-REGION
  - 8 deleted nucleotides at the 3' end of D-REGION
  - 3 deleted nucleotides at the 5' end of J-REGION
- "gc" content of N region(s)
  
  Ngc=2/6, it means that among the 6 added nucleotides during the N-diversity, 2 are C or G
- are there some P nucleotides?
  
  Yes, it is an adenine (A) downstream of the V-REGION
- CDR3-IMGT length
  
  The CDR3-IMGT is 11 amino acids long.
  Indeed, Cys 104 (2nd-CYS) and Trp/Phe 118 (J-TRP/J-PHE) are not included into the CDR3-IMGT.
  In contrast, they are part of the junction and mark the junction ends.

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Created:: 16/04/2007
Last updated:: 22/09/2023
Authors:: Amina Mrani, Céline Protat and Marie-Paule Lefranc
Editor:: Chantal Ginestoux

IMGT Education

V-J and V-D-J junctions analysis

Sequence analysis

Search in IMGT/LIGM-DB

Using IMGT/V-QUEST

Using IMGT/JunctionAnalysis