riot-na

RIOT - Rapid Immunoglobulin Overview Tool

Have some raw antibody sequences? Find matching germlines, perform numbering and get results in a familiar AIRR format!

RIOT supports both nucleotide and amino acid sequences as well as all major schemes: KABAT, CHOTHIA, MARTIN and IMGT.

MOTIVATION

Antibodies are a cornerstone of the immune system, playing a pivotal role in identifying and neutralizing infections caused by bacteria, viruses, and other pathogens. Understanding their structure, function, can provide insights into both the body's natural defenses and the principles behind many therapeutic interventions, including vaccines and antibody-based drugs. The analysis and annotation of antibody sequences, including the identification of variable, diversity, joining, and constant genes, as well as the delineation of framework regions and complementarity determining regions, are essential for understanding their structure and function. Currently analyzing large volumes of antibody sequences for is routine in antibody discovery, requiring fast and accurate tools. While there are existing tools designed for the annotation and numbering of antibody sequences, they often have limitations such as being restricted to either nucleotide or amino acid sequences, reliance on non-uniform germline databases, or slow execution times. Here we present Rapid Immunoglobulin Overview Tool (RIOT), a novel open source solution for antibody numbering that addresses these shortcomings. RIOT handles nucleotide and amino acid sequence processing, comes with a free germline database, and is computationally efficient. We hope the tool will facilitate rapid annotation of antibody sequencing outputs for the benefit of understanding of antibody biology and discovering novel therapeutics.

Links:

• PyPI
• Source code
• Collab example

Requirements

• Python ^3.10

Quickstart

> pip install riot-na

> riot_na -s GGGCGTTTTGGCAC...

{
    "sequence_header": "-",
    "sequence": "GGGCGTTTTGGCAC...",
    "numbering_scheme": "imgt",
    "locus": "igh",
    "stop_codon": False,
    "vj_in_frame": True,
    "v_frameshift": False,
    "j_frameshift": False,
    "productive": True,
    "rev_comp": False,
    "complete_vdj": True,
    "v_call": "IGHV1-69*01",
    "d_call": "IGHD3-3*01",
    "j_call": "IGHJ6*02",
    "c_call": "IGHM",
    "v_frame": 0,
    ...
}

Installation

Riot is distributed in prebuild binary wheels for all major platforms. Just run in your chosen virtualenv:

pip install riot-na

Usage

CLI

Usage: riot_na [OPTIONS]

Options:
  -f, --input-file PATH           Path to input FASTA file.
  -s, --sequence TEXT             Input sequence.
  -o, --output-file PATH          Path to output CSV file. If not specified,
                                  stdout is used.
  --scheme [kabat|chothia|imgt|martin]
                                  Which numbering scheme should be used: IMGT,
                                  KABAT, CHOTHIA, MARTIN. Default IMGT
  --species [human|mouse]         Which species germline sequences should be
                                  used. Default is all species.
  --input-type [nt|aa]            What kind of sequences are provided on
                                  input. Default is nucleotide sequences.
  -p, --ncpu INTEGER              Number of parallel processes to use. Default
                                  is number of physical cores.
  -e, --extend_alignment BOOLEAN  Include unaligned beginning of the query
                                  sequence in numbering.This option impacts
                                  only amino acid sequences passed with -s option.
  --help                          Show this message and exit.

Examples:

Run on single sequence and print output to stdout:

riot_na -s <sequence>

Run on single sequence and save output to csv:

riot_na -s <sequence> -o result.csv

Run on fasta file:

riot_na -f input.fasta -o results.csv

API Nucleotides

from riot_na import create_riot_nt, Organism, Scheme, RiotNumberingNT, AirrRearrangementEntryNT

riot_nt: RiotNumberingNT = create_riot_nt(allowed_species = [Organism.HOMO_SAPIENS])
airr_result: AirrRearrangementEntryNT = riot_nt.run_on_sequence(
                    header = "SRR13857054.957936",
                    query_sequence = "GAACCAAACTGACTGTCCTAGGCCAGCCCAAGTCTTCGCCATCAGTCACCCTGTTTCCACCTTCCCCTGAAGAGCTAAAAAAA",
                    scheme = Scheme.KABAT
                )

API Amino Acids

from riot_na import create_riot_aa, Organism, Scheme, RiotNumberingAA, AirrRearrangementEntryAA

riot_aa: RiotNumberingAA = create_riot_aa(allowed_species = [Organism.HOMO_SAPIENS])
airr_result: AirrRearrangementEntryAA = riot_aa.run_on_sequence(
                    header = "SRR13385915.5101835",
                    query_sequence = "QVTLKESGPVLVKPTETLTLTCTVSGFSLSNARMGVSWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTISKDTSKSQVVLTMTNMDPGDTATYYCARRGGTIFGVVIILVRRPPL",
                    scheme = Scheme.KABAT,
                    extend_alignment = True
                )

Multiprocessing

Riot uses precompiled Rust module for prefiltering. This means the RiotNumberingNT/AA objects are unpickable, so you cannot pass it as a worker's parameter in eg. mp.Pool() or use it in Spark's UDF functions. There is however a simple way of achieving it, by using caching mechanism from cachetools package. Below you can find working and not working examples.

The following will not work:

import functools
import multiprocessing as mp
from riot_na import create_riot_aa, AirrRearrangementEntryNT, RiotNumberingAA

seqs = ["EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGGSFYYYYMDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTGHHHHHHHHG"] * 10

def worker(riot: RiotNumberingAA, seq: str) -> AirrRearrangementEntryNT:
    airr = riot.run_on_sequence("-", seq)
    return airr

riot = create_riot_aa()

worker_partial = functools.partial(worker, riot)

with mp.Pool() as pool:
    res = pool.map(worker_partial, seqs)

# Output: TypeError: cannot pickle 'builtins.Prefiltering' object

The proper way:

import multiprocessing as mp
from cachetools import cached
from riot_na import create_riot_aa, AirrRearrangementEntryNT

seqs = ["EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGGSFYYYYMDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTGHHHHHHHHG"] * 10

@cached(cache={})
def get_riot():
    return create_riot_aa()

def worker(seq: str) -> AirrRearrangementEntryNT:
    riot = get_riot()
    airr = riot.run_on_sequence("-", seq)
    return airr

with mp.Pool() as pool:
    res = pool.map(worker, seqs)

Spark UDF example:

import json
from pyspark.sql import SparkSession
from pyspark.sql.functions import udf
from cachetools import cached
from riot_na import create_riot_aa, RiotNumberingAA

spark = SparkSession.builder.appName("Riot on Spark").getOrCreate()

seq = "EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGGSFYYYYMDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTGHHHHHHHHG"

df = spark.createDataFrame([{"seq":  seq}]*10)

@cached(cache={})
def get_riot() -> RiotNumberingAA:
    return create_riot_aa()

@udf
def number_sequence(seq: str)-> str:
    riot = get_riot()
    airr = riot.run_on_sequence("-", seq)
    return json.dumps(airr.__dict__)

df.select(number_sequence("seq")).collect()

For a pure Python solution you can check riot_na/api/api_mp.py file. We are using this because we want to keep Riot's dependencies to a minimum.

Germline database

RIOT uses OGRDB as a primary source of germline alleles. Database version as of 22.01.2024 was used. C genes are imported from igblast FTP site fhttps://ftp.ncbi.nih.gov/blast/executables/igblast/release/database/ncbi_human_c_genes.tar

Data format

This section describes the fields of numbering result object AirrRearrangementEntry(AA). It is based on AIRR Rearrangement Schema format extended by 7 columns highlighted in the table (bold) and emptied of the unnecessary ones. There are also some differences in fields’ definitions, so the AIRR format specification should be treated only as a loose reference. Description of the original AIRR format can be found here.

Attributes:

1. All fields in the format are required (always present)
2. All fields are nullable, with the exception of sequence_header and sequence

AirrRearrangementNT fields definitions

Name	Type	Definition
sequence_header	string	Fasta header for given input sequence (when numbering a FASTA file) or value of sequence_header parameter (when using RiotNumberingNT API).
sequence	string	The query nucleotide sequence. Usually, this is the unmodified input sequence, but can be reverse complemented if needed.
sequence_aa	string	Translated query sequence.
numbering_scheme	enum ["imgt", "kabat", "chothia", "martin"]	Used numbering scheme, default is "imgt".
locus	string	Gene locus (chain type).
stop_codon	boolean	True if the aligned sequence contains a stop codon.
vj_in_frame	boolean	True if the V and J gene alignments are in-frame. In details: distance between v_alignement reading frame and j_alignment reading frame is divisible by 3.
v_frameshift	boolean	True if the V gene in the query nucleotide sequence contains a translational frameshift relative to the frame of the V gene reference sequence. In other words: sum of insertions and deletions between consecutive matches in alignment is divisible by 3.
j_frameshift	boolean	True if the J gene in the query nucleotide sequence contains a translational frameshift relative to the frame of the J gene reference sequence. In other words: sum of insertions and deletions between consecutive matches in alignment is divisible by 3.
productive	boolean	True if the V(D)J sequence is predicted to be productive. In details: stop_codon is False and vj_in_frame is True
rev_comp	boolean	True if the alignment is on the opposite strand (reverse complemented) with respect to the query sequence. If True, indicates the sequence field contains a reverse complemented original query sequence.
complete_vdj	boolean	True if the sequence alignment spans the entire V(D)J region. Meaning, sequence_alignment includes both the first V gene codon that encodes the mature polypeptide chain (i.e., after the leader sequence) and the last complete codon of the J gene (i.e., before the J-C splice site). This does not require an absence of deletions within the internal FWR and CDR regions of the alignment.
v_call	string	V gene with allele.
d_call	string	D gene with allele.
j_call	string	J gene with allele.
c_call	string	Constant region gene with allele.
v_frame	enum [0, 1, 2]	V frame offset from v_alignment_start.
j_frame	enum [0, 1, 2]	J frame offset from j_alignment_start.
sequence_alignment	string	Gapped alignment of query sequence spanning V-J segment aligned to germline, reverse complemented if needed.
germline_alignment	string	Gapped aligned germline sequence spanning the same region as the sequence_alignment field (V(D)J region). Segments between matched germlines are gapped to match query sequence length.
sequence_alignment_aa	string	Amino acid translation of the sequence_alignment.
germline_alignment_aa	string	Amino acid translation of the germline_alignment.
v_alignment_start	integer	Start position of the V gene alignment in sequence_alignment (1-based closed interval).
v_alignment_end	integer	End position of the V gene alignment in sequence_alignment (1-based closed interval).
d_alignment_start	integer	Start position of the D gene alignment in sequence_alignment (1-based closed interval).
d_alignment_end	integer	End position of the D gene alignment in sequence_alignment (1-based closed interval).
j_alignment_start	integer	Start position of the J gene alignment in sequence_alignment (1-based closed interval).
j_alignment_end	integer	End position of the J gene alignment in sequence_alignment (1-based closed interval).
c_alignment_start	integer	Start position of the C gene alignment in sequence_alignment (1-based closed interval).
c_alignment_end	integer	End position of the C gene alignment in sequence_alignment (1-based closed interval).
v_sequence_alignment	string	Aligned portion of query sequence assigned to the V gene.
v_sequence_alignment_aa	string	Amino acid translation of the v_sequence_alignment field.
v_germline_alignment	string	Aligned V gene germline sequence.
v_germline_alignment_aa	string	Aligned amino acid V gene germline sequence.
d_sequence_alignment	string	Aligned portion of query sequence assigned to the D gene.
d_germline_alignment	string	Aligned D gene germline sequence.
j_sequence_alignment	string	Aligned portion of query sequence assigned to the J gene.
j_sequence_alignment_aa	string	Amino acid translation of the j_sequence_alignment field.
j_germline_alignment	string	Aligned J gene germline sequence.
j_germline_alignment_aa	string	Aligned amino acid J gene germline sequence.
c_sequence_alignment	string	Aligned portion of query sequence assigned to the constant region.
c_germline_alignment	string	Aligned constant region germline sequence.
fwr1	string	Nucleotide sequence of the aligned FWR1 region.
fwr1_aa	string	Amino acid translation of the fwr1 field.
cdr1	string	Nucleotide sequence of the aligned CDR1 region.
cdr1_aa	string	Amino acid translation of the cdr1 field.
fwr2	string	Nucleotide sequence of the aligned FWR2 region.
fwr2_aa	string	Amino acid translation of the fwr2 field.
cdr2	string	Nucleotide sequence of the aligned CDR2 region.
cdr2_aa	string	Amino acid translation of the cdr2 field.
fwr3	string	Nucleotide sequence of the aligned FWR3 region.
fwr3_aa	string	Amino acid translation of the fwr3 field.
cdr3	string	Nucleotide sequence of the aligned CDR3 region.
cdr3_aa	string	Amino acid translation of the cdr3 field.
fwr4	string	Nucleotide sequence of the aligned FWR4 region.
fwr4_aa	string	Amino acid translation of the fwr4 field.
junction	string	Junction region nucleotide sequence, where the junction is defined as the CDR3 plus the two flanking conserved codons.
junction_aa	string	Amino acid translation of the junction.
junction_length	integer	Number of nucleotides in the junction sequence.
junction_aa_length	integer	Number of amino acids in the junction sequence.
v_score	number	Alignment score (Smith-Waterman) for the V gene.
d_score	number	Alignment score (Smith-Waterman) for the D gene alignment.
j_score	number	Alignment score (Smith-Waterman) for the J gene alignment.
c_score	number	Alignment score (Smith-Waterman) for the C gene alignment.
v_cigar	string	CIGAR string for the V gene alignment.
d_cigar	string	CIGAR string for the D gene alignment.
j_cigar	string	CIGAR string for the J gene alignment.
c_cigar	string	CIGAR string for the C gene alignment.
v_support	number	V gene alignment E-value. Note: Every value less than 1.4e-45 will appear as 0.0 (due to single-precision floating point standard limitation)
d_support	number	D gene alignment E-value. Note: Every value less than 1.4e-45 will appear as 0.0 (due to single-precision floating point standard limitation)
j_support	number	J gene alignment E-value. Note: Every value less than 1.4e-45 will appear as 0.0 (due to single-precision floating point standard limitation)
c_support	number	C gene alignment E-value. Note: Every value less than 1.4e-45 will appear as 0.0 (due to single-precision floating point standard limitation)
v_identity	number	Fractional identity for the V gene alignment.
d_identity	number	Fractional identity for the D gene alignment.
j_identity	number	Fractional identity for the J gene alignment.
c_identity	number	Fractional identity for the C gene alignment.
v_sequence_start	integer	Start position of the V gene in the query sequence (1-based closed interval).
v_sequence_end	integer	End position of the V gene in the query sequence (1-based closed interval).
d_sequence_start	integer	Start position of the D gene in the query sequence (1-based closed interval).
d_sequence_end	integer	End position of the D gene in the query sequence (1-based closed interval).
j_sequence_start	integer	Start position of the J gene in the query sequence (1-based closed interval).
j_sequence_end	integer	End position of the J gene in the query sequence (1-based closed interval).
c_sequence_start	integer	Start position of the C gene in the query sequence (1-based closed interval).
c_sequence_end	integer	End position of the C gene in the query sequence (1-based closed interval).
v_germline_start	integer	Alignment start position in the V gene reference sequence (1-based closed interval).
v_germline_end	integer	Alignment end position in the V gene reference sequence (1-based closed interval).
d_germline_start	integer	Alignment start position in the D gene reference sequence (1-based closed interval).
d_germline_end	integer	Alignment end position in the D gene reference sequence (1-based closed interval).
j_germline_start	integer	Alignment start position in the J gene reference sequence (1-based closed interval).
j_germline_end	integer	Alignment end position in the J gene reference sequence (1-based closed interval).
c_germline_start	integer	Alignment start position in the C gene reference sequence (1-based closed interval).
c_germline_end	integer	Alignment end position in the C gene reference sequence (1-based closed interval).
fwr1_start	integer	FWR1 start position in the query sequence (1-based closed interval).
fwr1_end	integer	FWR1 end position in the query sequence (1-based closed interval).
cdr1_start	integer	CDR1 start position in the query sequence (1-based closed interval).
cdr1_end	integer	CDR1 end position in the query sequence (1-based closed interval).
fwr2_start	integer	FWR2 start position in the query sequence (1-based closed interval).
fwr2_end	integer	FWR2 end position in the query sequence (1-based closed interval).
cdr2_start	integer	CDR2 start position in the query sequence (1-based closed interval).
cdr2_end	integer	CDR2 end position in the query sequence (1-based closed interval).
fwr3_start	integer	FWR3 start position in the query sequence (1-based closed interval).
fwr3_end	integer	FWR3 end position in the query sequence (1-based closed interval).
cdr3_start	integer	CDR3 start position in the query sequence (1-based closed interval).
cdr3_end	integer	CDR3 end position in the query sequence (1-based closed interval).
fwr4_start	integer	FWR4 start position in the query sequence (1-based closed interval).
fwr4_end	integer	FWR4 end position in the query sequence (1-based closed interval).
sequence_aa_scheme_cigar	string	CIGAR string defining sequence_aa to scheme alignment.
scheme_residue_mapping	json string	Scheme numbering of sequence_alignment_aa - positions not present in this sequence are not included.
positional_scheme_mapping	json string	Mapping from absolute residue position in sequence_alignment_aa (0-based) to corresponding scheme position.
exc	string	Exception (if any) thrown during ANARCI numbering.
additional_validation_flags	json string	JSON string containing additional validation flags.

Additional validation flags

Following table describes additional validation flags calculated alongside main fields. Last 5 flags regarding conserved residues apply only then using IMGT schema.

Field name	AIRR fields required for calculation	Description
regions_in_aligned_sequence	all regions (fwr1, cdr1, fwr2 …); sequence_alignment	True if all region sequences, concatenated, are present in sequence_alignment.
regions_aa_in_aligned_sequence_aa	all _aa (fwr1_aa, cdr1_aa, …); sequence_alignment_aa	True if all region_aa sequences, concatenated, are present in sequence_alignment_aa.
translated_regions_in_aligned_sequence_aa	all regions (fwr1, cdr1, fwr2 …); sequence_alignment_aa; v_frame	True if all region sequences, concatenated and translated using v_frame, are present in sequence_alignment_aa.
correct_vj_in_frame	v_alignment_start; v_frame; j_alignment_start; j_frame	True if vj_in_frame is equal to: distance between v_alignement translation frame and j_alignment translation frame is divisible by 3.
cdr3_in_junction	cdr3; junction; cdr3_aa; junction_aa	True if cdr3 is present in junction and cdr3_aa is present in junction_aa.
locus_as_in_v_gene	locus; v_call	True if locus is consistent with the one specified in V gene (v_call).
v_gene_alignment	sequence; v_sequence_start; v_sequence_end; v_sequence_alignment	True if v_sequence_alignment is equal to substring in sequence from position v_sequence_start to v_sequence_end.
j_gene_alignment	sequence; j_sequence_start; j_sequence_end; j_sequence_alignment	True if j_sequence_alignment is equal to substring in sequence from position j_sequence_start to j_sequence_end.
c_gene_alignment	sequence; c_sequence_start; c_sequence_end; c_sequence_alignment	True if c_sequence_alignment is equal to substring in sequence from position c_sequence_start to c_sequence_end.
no_negative_offsets_inside_v_alignment	fwr1_start; fwr1_end; cdr1_start; cdr1_end; fwr2_start; fwr2_end; cdr2_start; cdr2_end; fwr3_start; fwr3_end; cdr3_start	True if there is no negative (missing) offset inside V alignment, eg.: fwr1_start == 1; fwr1_end == 35; cdr1_start == -1; cdr1_end == 65.
no_negative_offsets_inside_j_alignment	cdr3_end; fwr4_start; fwr4_end	True if there is no negative (missing) offset inside J alignment, eg.: cdr3_end == 293; fwr4_start == -1; fwr4_end == 326.
consecutive_offsets	all _start and _end	True if consecutive region_start and region_end offsets are ascendant, and no region_start is greater than corresponding region_end.
no_empty_cdr3	cdr3	True if cdr3 is present.
primary_sequence_in_sequence_alignment_aa	sequence_alignment_aa; scheme_residue_mapping	True if concatenation of scheme_residue_mapping amino acids results in a sequence that is a part of sequence_alignment_aa and amino acids are in correct order.
no_insertion_next_to_deletion_aa	sequence_aa_scheme_cigar	True if there are no insertions next to deletions - indicates correct CIGARs merging process.
insertions_in_correct_places	scheme_residue_mapping; numbering_scheme; locus	True if insertions are on schema-allowed positions.
correct_fwr1_offsets	sequence; v_sequence_start; fwr1_start; fwr1_end; fwr1	True if fwr1 is equal to substring in sequence cut from position fwr1_start up to fwr1_end. If fwr1_start is -1 (missing), v_sequence_start is used as a starting offset instead.
correct_cdr1_offsets	sequence; cdr1_start; cdr1_end; cdr1	True if cdr1 is equal to substring in sequence cut from position cdr1_start up to cdr1_end.
correct_fwr2_offsets	sequence; fwr2_start; fwr2_end; fwr2	True if fwr2 is equal to substring in sequence cut from position fwr2_start up to fwr2_end.
correct_cdr2_offsets	sequence; cdr2_start; cdr2_end; cdr2	True if cdr2 is equal to substring in sequence cut from position cdr2_start up to cdr2_end.
correct_fwr3_offsets	sequence; fwr3_start; fwr3_end; fwr3	True if fwr3 is equal to substring in sequence cut from position fwr3_start up to fwr3_end.
correct_cdr3_offsets	sequence; cdr3_start; cdr3_end; cdr3	True if cdr3 is equal to substring in sequence cut from position cdr3_start up to cdr3_end.
correct_fwr4_offsets	sequence; j_sequence_end; fwr4_start; fwr4_end; fwr4	True if fwr4 is equal to substring in sequence cut from position fwr4_start up to fwr4_end. If fwr4_end is -1 (missing), j_sequence_end is used as an ending offset instead.
no_empty_fwr1_in_v	v_sequence_alignment; fwr1	True if fwr1 is present.
no_empty_cdr1_in_v	v_sequence_alignment; cdr1	True if cdr1 is present.
no_empty_fwr2_in_v	v_sequence_alignment; fwr2	True if fwr2 is present.
no_empty_cdr2_in_v	v_sequence_alignment; cdr2	True if cdr2 is present.
no_empty_fwr3_in_v	v_sequence_alignment; fwr3	True if fwr3 is present.
no_empty_fwr4_in_j	j_sequence_alignment; fwr4	True if fwr4 is present.
conserved_C23_present	imgt_residue_mapping	True if conserved Cysteine on IMGT position 23 is present.
conserved_W41_present	imgt_residue_mapping	True if conserved Tryptophan on IMGT position 41 is present.
conserved_C104_present	imgt_residue_mapping	True if conserved Cysteine on IMGT position 104 is present.
conserved_W118_heavy_present	imgt_residue_mapping	True if conserved Tryptophan on IMGT position 118 is present (heavy chain only).
conserved_F118_light_present	imgt_residue_mapping	True if conserved Phenylalanine on IMGT position 118 is present (light chain only).

AirrRearrangementAA field definitions

Airr data format was developed for nucleotide sequences. For the amino acid pipeline a similar to format was created. Most fields are analogous to nucleotide-based one, with _aa suffix in name.

Name	Type	Definition
sequence_header	string	Fasta header for given input sequence (when numbering a FASTA file) or value of sequence_header parameter (when using RiotNumberingNT API).
sequence_aa	string	The query sequence.
numbering_scheme	enum ["imgt", "kabat", "chothia", "martin"]	Used numbering scheme, default is "imgt".
locus	string	Gene locus (chain type).
stop_codon	boolean	True if the aligned sequence contains a stop codon.
productive	boolean	True if the V(D)J sequence is predicted to be productive. In details: stop_codon is False and V and J genes are detected.
complete_vdj	boolean	True if the sequence alignment spans the entire V(D)J region. Meaning, sequence alignment includes both the first V amino acid and the last of the J gene (i.e., before the J-C splice site). This does not require an absence of deletions within the internal FWR and CDR regions of the alignment.
v_call	string	V gene with allele.
j_call	string	J gene with allele.
germline_alignment_aa	string	Assembled, aligned, full-length inferred germline sequence spanning the same region as the sequence_alignment_aa field (V-J region).
sequence_alignment_aa	string	Segment of query sequence spanning V-J aligned to germline.
v_alignment_start_aa	integer	Start position of the V gene alignment in sequence_alignment_aa (1-based closed interval).
v_alignment_end_aa	integer	End position of the V gene alignment in sequence_alignment_aa (1-based closed interval).
j_alignment_start_aa	integer	Start position of the J gene alignment in sequence_alignment_aa (1-based closed interval).
j_alignment_end_aa	integer	End position of the J gene alignment in sequence_alignment_aa (1-based closed interval).
v_sequence_alignment_aa	string	Aligned portion of query sequence assigned to the V gene.
v_germline_alignment_aa	string	Aligned V gene germline sequence.
j_sequence_alignment_aa	string	Aligned portion of query sequence assigned to the J gene.
j_germline_alignment_aa	string	Aligned J gene germline sequence.
fwr1_aa	string	Amino acid sequence of the aligned FWR1 region.
cdr1_aa	string	Amino acid sequence of the aligned CDR1 region.
fwr2_aa	string	Amino acid sequence of the aligned FWR2 region.
cdr2_aa	string	Amino acid sequence of the aligned CDR2 region.
fwr3_aa	string	Amino acid sequence of the aligned FWR3 region.
cdr3_aa	string	Amino acid sequence of the aligned CDR3 region.
fwr4_aa	string	Amino acid sequence of the aligned FWR4 region.
junction_aa	string	Junction region nucleotide sequence, where the junction is defined as the CDR3 plus the two flanking conserved amino acids.
junction_aa_length	integer	Number of amino acids in the junction sequence.
v_score_aa	number	Alignment score (Smith-Waterman) for the V gene.
j_score_aa	number	Alignment score (Smith-Waterman) for the J gene alignment.
v_cigar_aa	string	CIGAR string for the V gene alignment.
j_cigar_aa	string	CIGAR string for the J gene alignment.
v_support_aa	number	V gene alignment E-value. Note: Every value less than 1.4e-45 will appear as 0.0 (due to single-precision floating point standard limitation)
j_support_aa	number	J gene alignment E-value. Note: Every value less than 1.4e-45 will appear as 0.0 (due to single-precision floating point standard limitation)
v_identity_aa	number	Fractional identity for the V gene alignment.
j_identity_aa	number	Fractional identity for the J gene alignment.
v_sequence_start_aa	integer	Start position of the V gene in the query sequence (1-based closed interval).
v_sequence_end_aa	integer	End position of the V gene in the query sequence (1-based closed interval).
j_sequence_start_aa	integer	Start position of the J gene in the query sequence (1-based closed interval).
j_sequence_end_aa	integer	End position of the J gene in the query sequence (1-based closed interval).
v_germline_start_aa	integer	Alignment start position in the V gene reference sequence (1-based closed interval).
v_germline_end_aa	integer	Alignment end position in the V gene reference sequence (1-based closed interval).
j_germline_start_aa	integer	Alignment start position in the J gene reference sequence (1-based closed interval).
j_germline_end_aa	integer	Alignment end position in the J gene reference sequence (1-based closed interval).
fwr1_start_aa	integer	FWR1 start position in the query sequence (1-based closed interval).
fwr1_end_aa	integer	FWR1 end position in the query sequence (1-based closed interval).
cdr1_start_aa	integer	CDR1 start position in the query sequence (1-based closed interval).
cdr1_end_aa	integer	CDR1 end position in the query sequence (1-based closed interval).
fwr2_start_aa	integer	FWR2 start position in the query sequence (1-based closed interval).
fwr2_end_aa	integer	FWR2 end position in the query sequence (1-based closed interval).
cdr2_start_aa	integer	CDR2 start position in the query sequence (1-based closed interval).
cdr2_end_aa	integer	CDR2 end position in the query sequence (1-based closed interval).
fwr3_start_aa	integer	FWR3 start position in the query sequence (1-based closed interval).
fwr3_end_aa	integer	FWR3 end position in the query sequence (1-based closed interval).
cdr3_start_aa	integer	CDR3 start position in the query sequence (1-based closed interval).
cdr3_end_aa	integer	CDR3 end position in the query sequence (1-based closed interval).
fwr4_start_aa	integer	FWR4 start position in the query sequence (1-based closed interval).
fwr4_end_aa	integer	FWR4 end position in the query sequence (1-based closed interval).
sequence_aa_scheme_cigar	string	CIGAR string defining sequence_alignment_aa to scheme alignment.
scheme_residue_mapping	json string	Scheme numbering of sequence_alignment_aa - positions not present in this sequence are not included.
positional_scheme_mapping	json string	Mapping from absolute residue position in sequence_alignment_aa (0-based) to corresponding scheme position.
exc	string	Exception (if any) thrown during ANARCI numbering.
additional_validation_flags	json string	JSON string containing additional validation flags.

Additional validation flags (AA)

Following table describes additional validation flags calculated alongside main fields. Last 5 flags regarding conserved residues apply only then using IMGT schema.

	AIRR fields required for calculation	Description
regions_aa_in_aligned_sequence_aa	all _aa (fwr1_aa_aa, cdr1_aa_aa, …); sequence_alignment_aa	True if all region_aa sequences, concatenated, are present in sequence_alignment_aa.
locus_as_in_v_gene	locus; v_call	True if locus is consistent with the one specified in V gene (v_call).
v_gene_alignment_aa	sequence; v_sequence_start_aa; v_sequence_end_aa; v_sequence_alignment_aa	True if v_sequence_alignment_aa is equal to substring in sequence from position v_sequence_start_aa to v_sequence_end_aa.
j_gene_alignment_aa	sequence; j_sequence_start_aa; j_sequence_end_aa; j_sequence_alignment_aa	True if j_sequence_alignment_aa is equal to substring in sequence from position j_sequence_start_aa to j_sequence_end_aa.
no_negative_offsets_inside_v_alignment_aa	fwr1_aa_start_aa; fwr1_aa_end_aa; cdr1_aa_start_aa; cdr1_aa_end_aa; fwr2_aa_start_aa; fwr2_aa_end_aa; cdr2_aa_start_aa; cdr2_aa_end_aa; fwr3_aa_start_aa; fwr3_aa_end_aa; cdr3_aa_start_aa	True if there is no negative (missing) offset inside V alignment, eg.: fwr1_aa_start_aa == 1; fwr1_aa_end_aa == 26; cdr1_aa_start_aa == -1; cdr1_aa_end_aa == 38.
no_negative_offsets_inside_j_alignment_aa	cdr3_aa_end_aa; fwr4_aa_start_aa; fwr4_aa_end_aa	True if there is no negative (missing) offset inside J alignment, eg.: cdr3_aa_end_aa == 117; fwr4_aa_start_aa == -1; fwr4_aa_end_aa == 128.
consecutive_offsets_aa	all _start_aa and _end_aa	True if consecutive region_start_aa and region_end_aa offsets are ascendant, and no region_start_aa is greater than corresponding region_end_aa.
no_empty_cdr3_aa	cdr3_aa	True if cdr3_aa is present.
primary_sequence_in_sequence_alignment_aa	sequence_alignment_aa; scheme_residue_mapping	True if concatenation of scheme_residue_mapping amino acids results in a sequence that is a part of sequence_alignment_aa and amino acids are in correct order.
no_insertion_next_to_deletion_aa	sequence_aa_scheme_cigar	True if there are no insertions next to deletions - indicates correct CIGARs merging process.
insertions_in_correct_places	scheme_residue_mapping; numbering_scheme; locus	True if insertions are on schema-allowed positions.
correct_fwr1_aa_offsets	sequence; v_sequence_start_aa; fwr1_aa_start_aa; fwr1_aa_end_aa; fwr1_aa	True if fwr1_aa is equal to substring in sequence cut from position fwr1_aa_start_aa up to fwr1_aa_end_aa. If fwr1_aa_start_aa is -1 (missing), v_sequence_start_aa is used as a starting offset instead.
correct_cdr1_aa_offsets	sequence; cdr1_aa_start_aa; cdr1_aa_end_aa; cdr1_aa	True if cdr1_aa is equal to substring in sequence cut from position cdr1_aa_start_aa up to cdr1_aa_end_aa.
correct_fwr2_aa_offsets	sequence; fwr2_aa_start_aa; fwr2_aa_end_aa; fwr2_aa	True if fwr2_aa is equal to substring in sequence cut from position fwr2_aa_start_aa up to fwr2_aa_end_aa.
correct_cdr2_aa_offsets	sequence; cdr2_aa_start_aa; cdr2_aa_end_aa; cdr2_aa	True if cdr2_aa is equal to substring in sequence cut from position cdr2_aa_start_aa up to cdr2_aa_end_aa.
correct_fwr3_aa_offsets	sequence; fwr3_aa_start_aa; fwr3_aa_end_aa; fwr3_aa	True if fwr3_aa is equal to substring in sequence cut from position fwr3_aa_start_aa up to fwr3_aa_end_aa.
correct_cdr3_aa_offsets	sequence; cdr3_aa_start_aa; cdr3_aa_end_aa; cdr3_aa	True if cdr3_aa is equal to substring in sequence cut from position cdr3_aa_start_aa up to cdr3_aa_end_aa.
correct_fwr4_aa_offsets	sequence; j_sequence_end_aa; fwr4_aa_start_aa; fwr4_aa_end_aa; fwr4_aa	True if fwr4_aa is equal to substring in sequence cut from position fwr4_aa_start_aa up to fwr4_aa_end_aa. If fwr4_aa_end_aa is -1 (missing), j_sequence_end_aa is used as an ending offset instead.
no_empty_fwr1_aa_in_v	v_sequence_alignment_aa; fwr1_aa	True if fwr1_aa is present.
no_empty_cdr1_aa_in_v	v_sequence_alignment_aa; cdr1_aa	True if cdr1_aa is present.
no_empty_fwr2_aa_in_v	v_sequence_alignment_aa; fwr2_aa	True if fwr2_aa is present.
no_empty_cdr2_aa_in_v	v_sequence_alignment_aa; cdr2_aa	True if cdr2_aa is present.
no_empty_fwr3_aa_in_v	v_sequence_alignment_aa; fwr3_aa	True if fwr3_aa is present.
no_empty_fwr4_aa_in_j	j_sequence_alignment_aa; fwr4_aa	True if fwr4_aa is present.
conserved_C23_present	scheme_residue_mapping	True if conserved Cysteine on IMGT position 23 is present.
conserved_W41_present	scheme_residue_mapping	True if conserved Tryptophan on IMGT position 41 is present.
conserved_C104_present	scheme_residue_mapping	True if conserved Cysteine on IMGT position 104 is present.
conserved_W118_heavy_present	scheme_residue_mapping	True if conserved Tryptophan on IMGT position 118 is present (heavy chain only).
conserved_F118_light_present	scheme_residue_mapping	True if conserved Phenylalanine on IMGT position 118 is present (light chain only).

Examples

Sample usage of the software is presented at https://colab.research.google.com/drive/1xKO4udsX5gmnY88eDKWsQaUnHsLuFwVA?usp=sharing. To give users the ability to use RIOT with a custom database, we provide google colab script which showcases how to build a custom germline database for RIOT. It is available at https://colab.research.google.com/drive/1VCStUKgZ1ggi2Xf5YV7hFWHxxzP29BjK?usp=sharing.

Development

RIOT uses prefiltering module written in Rust, which requires some extra steps to install from source.

# Install Poetry

curl -sSL https://install.python-poetry.org | python3 - --version 1.7.1

# Add `export PATH="/root/.local/bin:$PATH"` to your shell configuration file.****

# Download and run the Rust installation script
curl --proto '=https' --tlsv1.2 -sSf https://sh.rustup.rs | sh -s -- -y

# Restart shell to reload PATH

# Verify the installation
!poetry --version
!rustc --version
!cargo --version

git clone https://github.com/NaturalAntibody/riot_na
cd riot_na

poetry install
poetry run maturin develop -r
poetry install

Citing this work

The code and data in this package is based on the following paper <we release the paper once it clears peer review>. If you use it, please cite:

@misc{riot,
      title={RIOT - Rapid Immunoglobulin Overview Tool - rapid annotation of nucleotide and amino acid immunoglobulin sequences using an open germline database.},
      author={Paweł Dudzic, Bartosz Janusz, Tadeusz Satława, Dawid Chomicz, Tomasz Gawłowski, Rafał Grabowski, Przemysław Jóźwiak, Mateusz Tarkowski, Maciej Mycielski, Sonia Wróbel, Konrad Krawczyk*},

}