Computational protocol: Multi Step Selection in Ig H Chains is Initially Focused on CDR3 and Then on Other CDR Regions

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Protocol publication

[…] Sequences were grouped into clones using a two-step approach. First, the germline VH and JH of each sequence were determined by aligning all possible germline VH and JH (based on the IMGT germline library) () against the sequence using the Basic Local Alignment Search Tool (BLAST) ().Next, in order to count the clones, we grouped all sequences according to their VH and JH usage as well as the distance between VH and JH, since SHMs usually do not produce additions or deletions of nucleotides. Thus, every clone emerging from the same founder cell should have the same distance between VH and JH. We then took all of the sequences with the same VH, JH, and distance between VH and JH and grouped them using a phylogenic approach. The distance between VH and JH was computed by positioning the IMGT germline VH and JH genes on the observed sequence and determining the distance between the last nucleotide of VH and the first nucleotide of JH.All the sequences with equal VH, JH, and distance were aligned together with an artificial sequence composed of the germline and gaps between them. Within each group, the sequences were aligned (using MUSCLE 3.6) (), and a phylogenetic tree was built using maximum parsimony () and/or neighbor joining () methods (from the PHYLIP 3.69 program package). We then parsed this tree with a cutoff distance of four mutations into clones. Thus, a clone was defined as a set of sequences that are similar one to each other, up to a distance of four mutations. […]

Pipeline specifications

Software tools BLASTN, MUSCLE, PHYLIP
Databases IMGT
Applications Phylogenetics, Nucleotide sequence alignment