ANARCI: antigen receptor numbering and receptor classification

1The variable domains of antibodies and T-cell receptors (TCR) contain these proteins’ major binding regions. Alignment of these variable sequences to a numbering scheme allows equivalent residue positions to be annotated and for different molecules to be compared. Performing numbering is fundamental for immunoinformatics analysis and rational engineering of therapeutic molecules.

2Several numbering schemes have been proposed, each is favoured by scientists in different immunological disciplines. The Kabat scheme was developed based on the location of regions of high sequence variation between sequences of the same domain type. It numbers antibody heavy (VH) and light (Vλ and Vκ) variable domains differently. Chothia’s scheme is the same as Kabat’s but corrects where an insertion is annotated around the first VH complementarity determining region (CDR) so that it corresponds to a structural loop. Similarly, the Enhanced Chothia scheme makes further structural corrections of indel positions.

3In contrast to these Kabat-like schemes, IMGT and AHo both define unique schemes for antibody and T cell receptor (TCR) (Vα and Vβ) variable domains. Thus, equivalent residue positions can easily be compared between domain types. IMGT and AHo differ in the number of positions they annotate (128 and 149 respectively) and where they consider indels to occur.

4Separate online interfaces exist that can apply each numbering scheme: Kabat, Chothia and Enhanced Chothia through Abnum; IMGT through DomainGapAlign; and AHo through PyIgClassify. No program currently exists that can apply all schemes or for which an executable is available under open license.

5We have developed ANARCI, a program that can annotate sequences with all five of the numbering schemes described above. We provide both a web-interface and the software under open license so that these fundamental annotations can be easily available for further immunoinformatics analyses.

6ANARCI takes single or multiple amino-acid protein sequences as input. The program aligns each sequence to a set of Hidden Markov Models (HMMs) using HMMER3. Each HMM describes the putative germ-line sequences for a domain type (VH, Vλ or Vκ, Vα or Vβ) of a particular species (Human, Mouse, Rat, Rabbit, Pig or Rhesus Monkey). The most significant alignment is then used to apply one of five numbering schemes.

7The HMM for each domain type from each species was built in the following way: The pre-aligned (gapped) germ-line sequences for the v-gene segment of each available species and domain type were downloaded from the IMGT/Gene Database. The sequences of the j-gene segment were also downloaded. These were aligned to a single reference sequence using Muscle with a large (−10) gap-open penalty.All possible pairwise combinations of the relevant v and j gene segments were taken to form a set of putative germ-line domain sequences. For the VH domain, the d gene segment was not included. Each position in the alignment represents one of the 128 positions in the IMGT numbering scheme.From the alignment an HMM is built using the hmmbuild tool. Here, the ‘—hand’ option is specified to preserve the structure of the alignment.

8The pre-aligned (gapped) germ-line sequences for the v-gene segment of each available species and domain type were downloaded from the IMGT/Gene Database. The sequences of the j-gene segment were also downloaded. These were aligned to a single reference sequence using Muscle with a large (−10) gap-open penalty.

9All possible pairwise combinations of the relevant v and j gene segments were taken to form a set of putative germ-line domain sequences. For the VH domain, the d gene segment was not included. Each position in the alignment represents one of the 128 positions in the IMGT numbering scheme.

10From the alignment an HMM is built using the hmmbuild tool. Here, the ‘—hand’ option is specified to preserve the structure of the alignment.

11In total, 24 HMMs were built describing variable domain types from six different species. These HMMs were combined into a single HMM database using hmmpress.

12An input sequence is aligned to each HMM using hmmscan. If an alignment has a bit-score of less than 100 it is not considered further. This threshold proves effective at preventing the false recognition of other IG-like proteins. Otherwise, the most significant alignment classifies its domain type and the alignment is translated into a chosen numbering scheme.

13ANARCI can apply the Kabat, Chothia, Extended Chothia, IMGT or AHo schemes to VH, Vλ and Vκ domain sequences. The IMGT and AHo schemes can also be applied to Vα and Vβ domain sequences. Where possible, a position in the HMM alignment is annotated with the equivalent position in the numbering scheme. In regions where there is no direct equivalence between the alignment and the numbering scheme the sequence is numbered according to the specification described in the corresponding publication. For example, HMM alignment position 40 for a VH sequence is equivalent to Kabat position 31-35X depending on the length of CDRH1.

14For each numbered domain a header is written that describes the most significant alignment including the species, chain type and alignment range. The numbering follows in a column delimited format. Alternatively, users may import ANARCI as a Python module and use the API within their own scripts.

15With the rise of next generation sequencing, the ability to annotate large numbers of antibody sequences is becoming a common task. We used ANARCI to number a set of 1 936 119 VH sequences taken from a vaccination response study performed at Oxford University. The algorithm took three hours wall clock time using 32-cores with AMD Opteron 6272 Processors. All but 9560 sequences were successfully numbered. Where numbering failed the sequences had very unusual insertions or deletions that may be a result of sequencing errors.

16In addition to the command line tool we provide a webserver interface to ANARCI (Fig. 1). Users may submit a single amino-acid sequence or a Fasta file of multiple sequences to apply their chosen scheme. The interface displays the assigned species and type of domain, the location of each domain in the sequence and, using the JSAV library, the annotated numbering scheme. Plain text or CSV formatted output files are available for download.

17We have developed ANARCI, a program for annotating antigen receptor variable domain amino-acid sequences with five commonly used numbering schemes. The program can be run as command-line tool or imported as a Python module for incorporation in custom scripts. We also provide a public web-browser interface that can annotate small numbers of sequences. ANARCI is freely distributed under the GPLv3 license and available at opig.stats.ox.ac.uk/webapps/anarci.