HLA Typing for the Next Generation
Neema P. Mayor, James Robinson, Alasdair McWhinnie, Swati Ranade, Kevin Eng, William Midwinter et al. · 2015 · 159 citationsRead the paper
Allele-level resolution data at primary HLA typing is the ideal for most histocompatibility testing laboratories. Many high-throughput molecular HLA typing approaches are unable to determine the phase of observed DNA sequence polymorphisms, leading to ambiguous results. The use of higher resolution methods is often restricted due to cost and time limitations. Here we report on the feasibility of using Pacific Biosciences' Single Molecule Real-Time (SMRT) DNA sequencing technology for high-resolution and high-throughput HLA typing. Seven DNA samples were typed for HLA-A, -B and -C. The results showed that SMRT DNA sequencing technology was able to generate sequences that spanned entire HLA Class I genes that allowed for accurate allele calling. Eight novel genomic HLA class I sequences were identified, four were novel alleles, three were confirmed as genomic sequence extensions and one corrected an existing genomic reference sequence. This method has the potential to revolutionize the field of HLA typing. The clinical impact of achieving this level of resolution HLA typing data is likely to considerable, particularly in applications such as organ and blood stem cell transplantation where matching donors and recipients for their HLA is of utmost importance.
What the full text shows
Working prototype- Absolute concordance between SMRT sequencing consensus sequences and expected/observed alleles after Sanger confirmation. — 100% (7 DNA samples typed for HLA-A, -B, and -C)
- Minimum depth of coverage achieved for all alleles. — >150x (median 462.5) (Post-processing stage of SMRT data analysis)
- Consensus sequences achieved high mean Quality Value (QV). — Mean QV 74.079 (range 71.937–80) (100% of total consensus sequences generated)
- Identification of novel genomic HLA class I sequences. — 8 novel sequences (4 novel alleles, 3 genomic extensions, 1 correction) (Comparison of SMRT results to IMGT/HLA Database)
- Accurate calling of alleles with notable non-coding deletions. (Samples containing B*73:01 and C*17:01)
- Setup
- 7 DNA samples (derived from blood and Oragene saliva) and 3 B-Lymphoblastoid Cell Lines (B-LCLs) tested for HLA-A, -B, and -C loci.
- How
- Full-length HLA Class I genes were amplified via PCR using TaKaRa LA DNA polymerase and sequenced using the Pacific Biosciences RS II SMRT DNA Sequencing System. SMRTbell adaptors were ligated to equimolar pools of amplicons, followed by sequencing with P4 enzyme and C2 chemistry.
- Limitations
- Allelic imbalance observed during PCR amplification, particularly for HLA-B. · Potential for loading bias or quantification errors due to amplicon concentrations being at the upper limit of the quantification kit (0.5–50 ng/μl). · Testing of homopolymer regions was limited to a maximum of 9-mers (T nucleotides).
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