HLA sequencing

PacBio highly accurate long-read sequencing of the Human Leukocyte Antigen (HLA) system provides complete and deep full gene HLA profiling without fragmentation. PacBio sequencing overcomes the high-sequence homology and hyper polymorphisms to identify true HLA allelic diversity.

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Access the full spectrum of polymorphisms across the HLA system

Highly accurate long reads – HiFi reads – with single-molecule resolution make Single Molecule, Real-Time (SMRT) Sequencing technology ideal for generating unambiguous, phase-resolved imputation-free HLA typing, so that you can:

  • Span complete HLA class I genes and long amplicons of HLA class II allowing for four field HLA genotyping
  • Fully phase polymorphisms with allele specificity across SNP-poor regions of HLA genes
  • Achieve unambiguous allele-level segregation without imputation
  • Detect variants in regulatory regions within 5’ UTRs, introns, and 3’ UTRs
  • Fully characterize minor variants in polyclonal samples, such as cancer or transcripts
  • Obtain direct evidence for new HLA alleles through de novo, reference-free consensus generation

From DNA to Imputation-free HLA Typing

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Library preparation

Fast and easy library preparation performed in a single tube.

  • Utilize off-the-shelf enrichment methods
  • Start with high-quality, nucleic acids, as low as 250 ng for a 250 bp amplicon
  • Create SMRTbell templates of amplicons of various sizes, from hundreds of bases to kilobase scale.
  • Optimize throughput with flexible barcoding options
    • Amplify PCR products using target-specific primers with incorporated barcodes
    • Add barcoded universal primers into amplicons via a simple 2-step PCR process
    • Attach barcoded overhang adapters during ligation without modifying existing primers
    • Multiplex up to 10,000 samples per SMRT Cell

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Sequencing

Use the Sequel systems to accurately sequence targets in the human genome.

  • Generate HiFi reads
    • Q20 single-molecule accuracy reads
    • Up to 4,000,000 reads per SMRT Cell 8M
  • Sequence to desired coverage based on project needs
    • Target 30-fold coverage for variant detection
    • Increase coverage for minor variant detection (~6,000-fold coverage for 1% sensitivity)

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Data analysis

Analysis solutions for every user in the lab with the PacBio analytical portfolio.

  • Fully characterize genetic complexity — structural variation, rare SNPs, indels, CNV, microsatellites, haplotypes, and phasing
  • Utilize a variety of analysis tools within SMRT Link
    • Generate HiFi reads (>Q20) using circular consensus sequencing (CCS) mode
    • Easily demultiplex barcodes within SMRT Link
    • Detect, quantitate, and phase single nucleotide polymorphisms within coding regions using minor variants analysis
    • Output data in standard file formats (BAM and FASTA/Q) for seamless integration with downstream analysis tools
  • Perform reference-free analysis of amplicons with pb amplicon analysis (pbaa)
  • HiFi reads are compatible with standard analysis tools for variant calling such as GATK and Deep Variant.
  • GenDx: NGSengine Software

Spotlight

Using SMRT Sequencing to achieve ultra high resolution for HLA matching on hemopoietic stem cell transplantation significantly improves 5-year overall survival

In a retrospective HLA typing study, SMRT Sequencing was used to establish 12/12 Ultra-High-Resolution (UHR) matching in a cohort of patients previously typed using other technologies. This study showed that better HLA matching was achieved when typing is done at UHR that includes exons outside of the antogen recognition domain, introns, and untranslated regions. The authors concluded that UHR matching can significantly improve outcomes for recipients of a volunteer unrelated donor hematopoietic cell transplantation for a hematologic malignancy and should be prospectively performed at donor selection. Explore this research further:

Mayor, N. P., et al. (2019). Recipients receiving better HLA-matched hematopoietic cell transplantation grafts, uncovered by a novel HLA typing method, have superior survival: A retrospective study. Biology of Blood and Marrow Transplantation, 25(3), 443–450.

Mayor_2019_Figure 1_UHR_HLA Typing

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