arcasHLA performs high resolution genotyping for HLA class I and class II genes from RNA sequencing, supporting both paired and single-end samples.

Make sure the following programs are in your PATH:

• Samtools
• bedtools
• pigz
• Kallisto v0.44.0
• Python 3.6

arcasHLA requires the following Python modules:

• Biopython
• NumPy
• Pandas

In order to test arcasHLA partial typing, we need to roll back the reference to an earlier version. First, fetch IMGT/HLA database version 3.24.0:

./arcasHLA reference --version 3.24.0

Extract reads:

./arcasHLA extract test/test.bam -o test/output --paired -t 8 -v

Complete typing:

./arcasHLA genotype test/output/test.extracted.1.fq.gz test/output/test.extracted.2.fq.gz -g A,B,C,DPB1,DQB1,DQA1,DRB1 -o test/output -t 8 -v

Expected output in test/output/test.genotype.json:

{"A": ["A*01:01:01", "A*03:01:01"], 
 "B": ["B*39:01:01", "B*07:02:01"], 
 "C": ["C*08:01:01", "C*01:02:01"], 
 "DPB1": ["DPB1*14:01:01", "DPB1*02:01:02"], 
 "DQA1": ["DQA1*02:01:01", "DQA1*05:03"], 
 "DQB1": ["DQB1*02:02:01", "DQB1*06:09:01"], 
 "DRB1": ["DRB1*10:01:01", "DRB1*14:02:01"]}

Partial typing:

./arcasHLA partial test/output/test.extracted.1.fq.gz test/output/test.extracted.2.fq.gz -g A,B,C,DPB1,DQB1,DQA1,DRB1 -G test/output/test.genotype.json -o test/output -t 8 -v

Expected output in test/output/test.partial_genotype.json:

{"A": ["A*01:01:01", "A*03:01:01"], 
 "B": ["B*07:02:01", "B*39:39:01"],
 "C": ["C*08:01:01", "C*01:02:01"], 
 "DPB1": ["DPB1*14:01:01", "DPB1*02:01:02"], 
 "DQA1": ["DQA1*02:01:01", "DQA1*05:03"], 
 "DQB1": ["DQB1*06:04:01", "DQB1*02:02:01"],
 "DRB1": ["DRB1*03:02:01", "DRB1*14:02:01"]}

Before further usage, remember to update to the current version.

./arcasHLA reference --update

To see the list of available tools, simply enter arcasHLA. To view the required and optional arguments for any of the tools enter arcasHLA [command] -h.

• extract : Extracts reads mapped to chromosome 6 and any HLA decoys or chromosome 6 alternates.
• genotype : Genotypes complete HLA alleles from extracted reads.
• partial : Genotypes partial HLA alleles from extracted reads and output from genotype.
• reference : Update, specify version or force rebuilding of HLA reference.
• merge : merge genotyping output for multiple samples into a single json file.

arcasHLA takes sorted BAM files and extracts chromosome 6 reads and related HLA sequences. If the BAM file is not indexed, this tool will run samtools index before extracting reads. By default, extract outputs a single FASTQ file; use the --paired flag for paired-end samples.

arcasHLA extract [options] /path/to/sample.bam 

Output: sample.1.fq.gz, sample.2.fq.gz

• --paired : paired-end reads (default: False)
• --unmapped : include unmapped reads, recommended if the aligner used marks multimapping reads as unmapped (default: False)
• --log FILE : log file for run summary (default: sample.extract.log)
• --o, --outdir DIR : output directory (default: .)
• --temp DIR : temp directory (default: /tmp)
• --keep_files : keep intermediate files (default: False)
• -t, --threads INT : number of threads (default: 1)
• -v, --verbose : verbosity (default: False)

To predict the most likely genotype (complete alleles), input the FASTQs produced by extract.

arcasHLA genotype [options] /path/to/sample.1.fq.gz /path/to/sample.2.fq.gz

Output: sample.alignment.p, sample.em.json, sample.genotype.json

If you have previously run genotype on a sample, you can run genotype again directly from sample.alignment.p to retype without aligning with Kallisto again. This is useful if you want to try different populations, genes and other parameters.

arcasHLA genotype [options] /path/to/sample.alignment.p

{'A': ['A*01:01:01', 'A*29:02:01'],
 'B': ['B*08:01:01', 'B*44:03:01'],
 'C': ['C*07:01:01', 'C*16:01:01'],
 'DQA1': ['DQA1*02:01:01', 'DQA1*05:01:01'],
 'DQB1': ['DQB1*02:01:01', 'DQB1*02:02:01'],
 'DRB1': ['DRB1*03:01:01', 'DRB1*07:01:01']}

• -g, --genes GENES : comma separated list of HLA genes (ex. A,B,C,DQA1,DQB1,DRB1)
• -p, --population POPULATION : sample population, options are asian_pacific_islander, black, caucasian, hispanic, native_american and prior (default: Prior)
• --tolerance FLOAT : convergence tolerance for transcript quantification (default: 10e-7)
• --max_iterations INT : maximmum number of iterations for transcript quantification (default: 1000)
• --drop_iterations INT : number of iterations before dropping low support alleles, a lower number of iterations is recommended for single-end and low read couunt samples (default: paired - 10, single - 4)
• --drop_threshold FLOAT : proportion of maximum abundance an allele needs to not be dropped (default: 0.1)
• --zygosity_threshold FLOAT : threshold for ratio of minor to major allele nonshared count to determine zygosity (default: 0.15)
• --log FILE : log file for run summary (default: sample.genotype.log)
• --o, --outdir DIR : output directory (default: .)
• --temp DIR : temp directory (default: /tmp)
• --keep_files : keep intermediate files (default: False)
• -t, --threads INT : number of threads (default: 1)
• -v, --verbose : verbosity (default: False)

Following genotyping, partial alleles can be predicted. This requires aligning the reads to an alternate, partial allele reference. The sample.genotype.json file from the previous step is required.

arcasHLA partial [options] -G /path/to/sample.genotype.json /path/to/sample.1.fq.gz /path/to/sample.2.fq.gz

Output: sample.partial_alignment.p, sample.partial_genotype.json

The options for partial typing are the same as complete. Partial typing, like complete, can be run from the intermediate alignment file.

To make analysis easier, this command will merge all jsons produced by genotyping. All .genotype.json files will be merged into a single run.genotypes.json file and all .partial_genotype.json files will be merged into run.partial_genotypes.json.

arcasHLA merge [options]

• --run RUN : run name
• --i, --indir DIR : input directory (default: .)
• --o, --outdir DIR : output directory (default: .)
• -v, --verbose : verbosity (default: False)

To update the reference to the latest IMGT/HLA version, run

arcasHLA reference --update

If you are running multiple tools to type HLAs, it can be helpful to use the same version of IMGT/HLA. You can select the version you like using the commithash from the IMGT/HLA Github.

arcasHLA reference --version [commithash]

If you suspect there is an issue with the reference files, rebuild the reference with the following command

arcasHLA reference --rebuild

• --update : update to latest IMGT/HLA version
• --version : checkout IMGT/HLA version using commithash
• --rebuild : rebuild HLA database
• -v, --verbose : verbosity (default: False)

R. Orenbuch, I. Filip, D. Comito, J. Shaman, I. Pe’er, and R. Rabadan. arcasHLA: high resolution HLA typing from RNA seq. bioRxiv doi: 10.1101/479824