Accurate Typing of Human Leukocyte Antigen (HLA) by Oxford Nanopore Sequencing
- The pipeline is validated to perform HLA typing using nanopore sequencing reads at 2-field resolution (limiting to the exons 2 and 3 of class I HLA genes at this point).
- Locus-specific sequencing reads should cover at least exons 2 and 3 of HLA-A, -B, and -C genes, and the number of reads is ideally above 1000 per locus.
The following software or compatible versions must be available in the $PATH:
- seqtk, version 1.0-r31
- BLASR, version 2.0.0
- Samtools, version 1.2 using htslib 1.2.1
- Bedtools, version 2.25.0
- Pyfaidx, version 0.4.8.3
- freebayes, version 1.1.0
- vcflib, version 1.0.0
- Blast, version 2.2.31
git clone https://github.com/cliu32/Athlon.git
cd Athlon
chmod +x athlon.sh
./athlon.sh <ReadNumber>
Three samples in the /data folder will be analyzed, each containing ~3000 reads for locus A, B, and C, respectively. When calling athlon.sh, please specify the number of reads (3000 or lower in this case) to be analyzed in the command line, for example:
./athlon.sh 1000
A new folder /data1000 will be generated to hold fastq files with randomly sampled reads from the original fastq files in the /data folder. Intermediate files during the analysis will be located the /rslt1000 folder. A .log file and a .rslt file will be generated in the /Athlon directory. The log file will document the process of selecting candidate alleles for each sample; the rslt file will report all the typing results at 3-field G-group level.
If you are starting from a fastq file, .fastq, that have not been demultiplexed, please follow these steps. i) Include the fastq file in the /rawdata folder, ii) update the primer sequences in the hla_primers_ABC.csv file in the /primer folder to match your primer sequences, and iii) run the following python script:
python kmer_split.py <fqName> hla_primers_ABC
Then copy all the demultiplexed fastq files to /data, which will be ready for athlon analysis. To customize the demultiplexing step, please follow the help message:
python kmer_split.py -h
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Copyright (c) 2018 Washington University Created by: Chang Liu
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