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Appendix

TCR/BCR refenrece sequences library

Default list and sequences of V, D, J and C genes used by MiXCR are taken from GenBank. Accession numbers of records used for each locus are listed in the following table:

+-----------------+-----------------+-------------+ +-----------------+-----------------+-------------+ | | | TRA/TRD | NG_001332.2 | | +-----------------+-------------+ | | TRB | NG_001333.2 | | +-----------------+-------------+ | | TRG | NG_001336.2 | | +-----------------+-------------+ | Homo sapiens | IGH | NG_001019.5 | | +-----------------+-------------+ | | IGK | NG_000834.1 | | +-----------------+-------------+ | | IGL | NG_000002.1 | +-----------------+-----------------+-------------+ +-----------------+-----------------+-------------+ | | | TRA/TRD | NG_007044.1 | | +-----------------+-------------+ | | TRB | NG_006980.1 | | +-----------------+-------------+ | | TRG | NG_007033.1 | | Mus musculus +-----------------+-------------+ | | IGH | NG_005838.1 | | +-----------------+-------------+ | | IGK | NG_005612.1 | | +-----------------+-------------+ | | IGL | NG_004051.1 | +-----------------+-----------------+-------------+

Alignment and mutations encoding

MiXCR outputs alignments in exportClones and exportAlignments as a list of 7 fields separated by | symbol as follows:

targetFrom | targetTo | targetLength | queryFrom | queryTo | mutations | alignmentScore

where

  • targetFrom - position of first aligned nucleotide in target sequence (sequence of gene feature from reference V, D, J or C gene used in alignment; e.g. VRegion in TRBV12-2); this boundary is inclusive
  • targetTo - next position after last aligned nucleotide in target sequence; this boundary is exclusive
  • targetLength - length of target sequence (e.g. length of VRegion in TRBV12-2)
  • queryFrom - position of first aligned nucleotide in query sequence (sequence of sequencing read or clonal sequence); this boundary is inclusive
  • queryTo - next position after last aligned nucleotide in query sequence; this boundary is exclusive
  • mutations - list of mutations from target sequence to query sequence (see below)
  • alignmentScore - score of alignment

all positions are zero-based (i.e. first nucleotide has index 0)

Mutations are encoded as a list of single-nucleotide edits (similar to what is used in definition of Levenshtein distance, i.e. insertions, deletions or substitutions); if one apply these mutations to aligned subsequence of target sequence, one will obtain aligned subsequence of query sequence.

Each single mutation (single-nucleotide edit) is encoded in the following way (without any spaces; some fields may absent in some cases, see description):

type [fromNucleotide] position [toNucleotide]

  • type of mutation (one letter):
  • S for substitution
  • D for deletion
  • I for insertion
  • fromNucleotide is a nucleotide in target sequence affected by mutation (applicable only for substitutions and deletions; absent for insertions)
  • position is a zero-based absolute position in target sequence affected by mutation; for insertions denotes position in target sequence right after inserted nucleotide
  • toNucleotide nucleotide after mutation (applicable only for substitutions and insertions; absent for deletions)

Note, that for deletions and substitutions

targetSequence[position] == fromNucleotide

i.e. target sequence always have fromNucleotide at position position; for insertions fromNucleotide field is absent

Here are several examples of single mutations:

  • SA4T - substitution of A at position 4 to T
  • DC12 - deletion of C at position 12
  • I15G - insertion of G before position 15

Consider the following BLAST-like alignments encoded in MiXCR notation:

  • Alignment without mutation

    target = TTGTGCTGACAGATACCCC
query  = CGAGTGCTGACAGATACCGTCGATGCT

BLAST like alignment:
2 GTGCTGACAGATACC 16
  |||||||||||||||
3 GTGCTGACAGATACC 17

MiXCR alignment:
2|17|19|3|18||75.0

subsequence from target (from nucleotide 0 to nucleotide 15) was found to be identical to susequence from query (from nucleotide 3 to nucleotide 18).

  • Alignment with mutation

    target = TTGTGCTGACAGATACCCC
query  = CGAGTGCTATAGACTACCGTCGATGCT

BLAST like alignment:
2 GTGCTGACAGA-TACC 16
  ||||| | ||| ||||
3 GTGCT-ATAGACTACC 17

MiXCR alignment:
2|17|19|3|18|DG7SC9TI13C|41.0

so, to obtain subseqeunce from query sequence from 3 to 18 we need to apply the following mutations to subsequence of target sequence from 2 to 16: - deletion of G at position 7 - substitution of C at position 9 to T - insertion of C before at position 13