Detailed example of clone detection
1. VH and JH detection
Raw sequences were aligned against all possible germline VH and JH from IgBLAST. Each
sequence was assigned the VH and JH segments that had the highest number of overlapping
nucleotides, assuming no deletion or insertion occurred. Since we did not attempt to identify
DH at this stage, we defined L as the distance between VH and JH by positioning the germline
VH and JH genes on the observed sequence and determining the distance between the end of
VH and the beginning of JH. See Figure.
Take for example the following sequences:
>1_11_10_8_27_34_11_8_GO4TH0H01ARBNQ rank=0093876 x=194.0 y=308.0
length=477;IGHV1-18 286 2 1;IGHD6-13 13 0 1;IGHJ6 53 0
1;CARVSSSSWHYYGMDVW;;IGHM 113 0 1
TGGTAACACAAACTATGCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGA
CACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACAC
GGCCGTGTATTACTGTGCGAGAGTGTCTAGCAGCAGCTGGCACTACTACGGT
>45_34_2_1_46_40_7_9_GO4TH0H01BQI53 rank=0047658 x=595.0 y=505.0
length=503;IGHV3-11 279 2 1;;IGHJ2 49 0 1;;;IGHM 99 1 1
TAGTATCATATACTACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGGGAC
AACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACG
GCCGTGCGAGAGACGATAGCAGTGGCTCCCACGACTATTGGTACTTCGATCTCT
…
V segment
L = Distance between V and J
J segment
For example, the first sequence was classified with V=1, J=10 and a distance of 10
nucleotides between them, and the second sequence with V=45 and J=2 and a distance of 13
nucleotides.
2. Sequence division into clones
In order to indentify sequences originating from the same clone, we first grouped all sequence
according to their VH and JH usage and L (the distance between V and J). Since SHM
occurring during clonal expansion are not supposed to induce deletion or addition, every
clone emerging from the same founder cell should have the same distance between VH and JH.
>1_L13
TGGTAACACAAACTATGCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGA
CACATCCACGAGCACAGCCTACATGGAGCCGAGGAGCCTGAGATCCGACGACAC
GGCCGCGTATTACTGTGCGAGAAGGGACGATTTTTGGAGTGGTTACTACGGT
>1_L15
TGGTAACACAAACTATGCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGA
CACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACAC
GGCCGTGTATTACTGTGCGAGAGTGGATAGTTCGGGGAGGTACTACTACGGT
>1_L16
TGGTAACACAAACTATGCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGA
CACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACAC
GGCCGTGTATTACTGTGCGAGAAGGGACGATTTTTGGAGTGGTTACTACGGT
>2_L25
TGGTAACACAAACTATGCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGA
CACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACAC
GGCCGTGTATTACTGTGCGAGAGACGCCGATTTTTGGAGTGGTTACTACGGT
…
>VJ
TGGTAACACAAACTATGCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGA
CACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACAC
GGCCGTGTATTACTGTGCGAGA----------ATTACTACTACTACTACGGT
For example, this group is constituted from sequences having V=1, J=10 and a
distance of 10 nucleotides between V and J germline sequences. Furthermore, the artificial
VJ sequence was built from the germline V=1 and J=10, and 10 gaps were added between
them.
All the sequences within a group were aligned together with an artificial sequence composed
of the germline sequences and gaps between them according to the distance between V and J
in a current group.
The sequences were aligned (using MUSCLE 3.6), and a phylogenetic tree was built using
one of the following methods (from PHYLIP 3.69 program package); maximum parsimony
for groups containing less than 100 sequences and neighbor joining otherwise. The resulting
trees were cut into independent trees at branches with more than 4 mutations between 2
sequences. Each sub-tree was defined as a clone.
For example, sequences L_16, L_13 and L_25 were classified as originating from the
same clone, whereas L_15 belongs to another clone.