ADVANCES IN DNA SEQUENCING TECHNOLOGIES FOR HIGH RESOLUTION HLA TYPING�Human Immunology, Volume 76, Issue 12, 2015

Low Hwei Yee

Scientific Officer

Seksyen of Histocompatibility & Immunogenetics

​

Introduction

• The introduction of PCR based amplification and sequencing of HLA Class I and Class II genes revealed a level of polymorphism far beyond the resolution of serologic or cellular based HLA typing.
• Now, the HLA community is still struggling to deal with this extensive and ever-expanding allelic diversity.

Number of alleles named by year from 1987 to the end of March 2019

Robinson J, Halliwell JA, Hayhurst JH, Flicek P, Parham P, Marsh SGE�The IPD and IMGT/HLA database: allele variant databases�Nucleic Acids Research (2015) 43:D423-431

​

Introduction

• The patchwork pattern of polymorphism identified by these initial sequencing studies was primarily localized to the exons that encoded the peptide binding domains of Class I and Class II molecules.
• The analysis of HLA polymorphism based on probe hybridization (SSOP) as well as on sequence-specific PCR priming (SSP) followed by amplicon detection (gel electrophoresis or fluorescence monitoring) focused on specific polymorphic sequence motifs in targeted amplicons.

Introduction

• The implemetation of Sanger based sequencing (SBT) of targeted HLA amplicons allowed the analysis of all the sequences within amplicons, giving rise to a high resolution typing method.

​

1998 – ABI 3700 Automated DNA Analyzer

2002 – ABI 3730x DNA Analyzer

2006 – Histogenetics established SBT

• Large volume SBT
• More than 3.8 millions samples were typed

Introduction

• This demands for high throughput, G group-level high resolution testing was mainly driven by unrelated donor registries around the world.
• Nonetheless, given the large number of HLA alleles, even SBT often yields ambiguous genotyping results, due to genomic regions not targeted by the primers and to the inability to set phase for linked polymorphisms within the amplicon for heterozygous samples.
• Next generation sequencing technologies were first commercially introduced in 2004 and they immediately revolutionized genomic and genetic research.

Introduction

• Several NGS systems are commercially available and each having its own strengths and weaknesses.

Illumina MiSeq

• Intergrates cluster generation, amplification, sequencing, and data processing into a single instrument.
• Sequencing by synthesis (SBS) technology.
• Based on reversible dye-terminator technology and engineered polymerases and is driving the development of high-throughput parallel sequencing technologies.

​

Pacific Biosciences

• Based on single molecule, real time (SMRT®) sequencingon the PacBio® RS II System.
• Captures information from SMRT® Cells containing 150,000 zero-mode waveguides that are simultaneously illuminated to detect the incorporation of phospholinked nucleotides by DNA polymerase.
• This method generates extraordinarily long reads of average length between 10 and 15 kb, with the top 5% reads >30 kb and the logest reads >60 kb.

Objective

• This paper describes Histogenetics’ experience in large-scale G group-level high resolution HLA typing using three different DNA sequencing platforms:
• ABI 3730 xl
• Illumina MiSeq
• PacBio RS II

Meterials and Methods

Amplification primer design

• Class I (A, B, C) and Class II (DRB1, DRB3, DRB4, DRB5, DQA1, DQB1, DPA1 and DPB1) were amplified by PCR with specific primers using genomic DNA as a template.
• Amplification primers, locus or group specific were designed according to the recommended read length for each sequencing platform.

Amplification Primer Design

Amplification Primer Design

Meterials and Methods

QIAamp 96 DNA Blood Kit (QIAGEN)

Meterials and Methods

Meterials and Methods

Results

• Since the introduction of SBT in 2006, over 4,695,352 specimens were typed as of May 21, 2015 by SBT.
• The majority of the specimens were volunteer donors from registries worldwide and including a small percentage of clinical specimens.
• Among those typed, 22,323 samples carried a new allele.

​

​

​

• Novel patterns that could not be sequenced in isolation were not submitted. They were found in approximately 6000 samples. Now they are being identified using PacBio.

Results

• The majority of the specimens were sequenced only for Antigen Recognition Site (ARS) encoding exons, which includes exon 2 and exon 3 for Class I genes and exon 2 for Class II genes. 🡪 G group-level high resolution typing
• Common and Well Defined (CWD) Null alleles were resolved by sequencing other exons that contain null mutations.
• Phasing of ARS exons for the ambiguous allele combination pairs that were produced by Sanger sequencing was accomplished with the use of allele- or group-specific sequencing primers to separate two alleles.

Results

• In March 2013, Histogenetics introduced a hybrid approach of Sanger plus Illumina MiSeq.
• A total 460,190 samples were typed with MiSeq plus Sanger to validate MiSeq data during the transition to NGS.
• During that period we developed and improved the primer pairs to achieve locus specific and short enough amplicons to run in the MiSeq platform (<500 bp, <250 bps each direction).

Results

• DNA sequencing results from MiSeq provide much higher resolution data compared to Sanger results for registry donor samples, and enable to provide 100% G group level high resolution typing for Class II and 97.75–99.94% for Class I. PacBio achieved 100% G group-level high resolution.

Results

• In October 2013, Illumina MiSeq was introduced as the primary method for high volume, G group-level high resolution HLA Typing.
• In October 2014, PacBio was introduced for HLA Class I typing for samples with ambiguities that are not resolved by other methods. Also PacBio was used to resolve novel alleles.

Results

• Besides pushing for higher volume typing, Histogenetics was also excelling in quality and accuracy with the strict quality control and quality assurance policy established in Histogenetics’ high throughput HLA typing process.
• National Marrow Donor Program (NMDP) is one of Histogenetics’ major clients, and has a strict quality control program where an average of 3% of blind QC samples are included in every batch of testing samples.

Discussion

• The challenges of molecular HLA typing laboratories are:
• fast turn-around time (TAT) for clinical specimens
• varying qualities and quantities of specimens
• different resolution requirements
• high volume typing for donor registries
• many other issues.
• Currently, a single DNA sequencing technology cannot meet all those requirements needed to deliver the results in a timely fashion.

Discussion

• Capillary Sanger technology
• gold DNA sequencing standard for a long time due to its high accuracy, simple chemistry, its highly automatable, scalable process and its fast sequencing run time.
• Drawback
• inability to phase the heterozygous sequences and the limited read lengths.

Discussion

• Next generation sequencing technologies (NGS)
• deliver single molecule sequencing
• Among others, Illumina chemistry has been most popular for genome, exome, and targeted sequencing for various applications.
• The most attractive features of Illumina sequencing are their high quality and high coverage of sequence data.
• The MiSeq platform has accomplished higher resolution HLA typing results and a faster and more cost effective and easier work flow compared to Sanger sequencing and other NGS platforms.
• Illumina chemistry provides error free long homopolymer (HP) reading

Discussion

• Drawback
• the sequencing read lengths and sequencing run time. Illumina DNA polymerase can read 250–300 bp in one direction, a maximum 600 bps pairedend reads; however, up to 100 bp of those reads are adopter, index and primer sequences, leaving 400–500 bp to sequence the target regions. This poses an exon phasing problem unless additional amplicons are introduced.
• missing insertions located in the forward and reverse sequence overlap region and long run times. Therefore it is not suitable for very fast TAT samples.

Discussion

• PacBio
• long read lengths with excellent phasing of the Exons and Introns and short run times.
• It produces high quality sequencing comparable to the other two technologies.
• It also provides us with an excellent alternative technology.
• Drawbacks
• a limitation in the barcoding (multiplexing) and a longer sample preparation time, but it compensates with longer sequence reads.
• could have error in long homopolymer reading (in low percentages).

Conclusion

• High resolution HLA matching reduces GVHD & improves overall patient survival after HSCT.
• All three sequencing technologies deliver high quality DNA sequence information, although each has its own limitations.
• However, when they are used to complement each other when necessary they revolutionized the way that we provide fast, affordable, and high quality HLA typing results.