June 1, 2021  |  

Genomic DNA sequences of HLA class I alleles generated using multiplexed barcodes and SMRT DNA Sequencing technology.

Allelic-level resolution HLA typing is known to improve survival prognoses post Unrelated Donor (UD) Haematopoietic Stem Cell Transplantation (HSCT). Currently, many commonly used HLA typing methodologies are limited either due to the fact that ambiguity cannot be resolved or that they are not amenable to high-throughput laboratories. Pacific Biosciences’ Single Molecule Real-Time (SMRT) DNA sequencing technology enables sequencing of single molecules in isolation and has read-length capabilities to enable whole gene sequencing for HLA. DNA barcode technology labels samples with unique identifiers that can be traced throughout the sequencing process. The use of DNA barcodes means that multiple samples can be sequenced in a single experiment but data can still be attributed to the correct sample. Here we describe the results of experiments that use DNA barcodes to facilitate sequencing of multiple samples for full-length HLA class I genes (known as multiplexing).


June 1, 2021  |  

HLA sequencing using SMRT Technology – High resolution and high throughput HLA genotyping in a clinical setting

Sequence based typing (SBT) is considered the gold standard method for HLA typing. Current SBT methods are rather laborious and are prone to phase ambiguity problems and genotyping uncertainties. As a result, the NGS community is rapidly seeking to remedy these challenges, to produce high resolution and high throughput HLA sequencing conducive to a clinical setting. Today, second generation NGS technologies are limited in their ability to yield full length HLA sequences required for adequate phasing and identification of novel alleles. Here we present the use of single molecule real time (SMRT) sequencing as a means of determining full length/long HLA sequences. Moreover we reveal the scalability of this method through multiplexing approches and determine HLA genotyping calls through the use of third party Gendx NGSengine® software.


June 1, 2021  |  

Long Amplicon Analysis: Highly accurate, full-length, phased, allele-resolved gene sequences from multiplexed SMRT Sequencing data.

The correct phasing of genetic variations is a key challenge for many applications of DNA sequencing. Allele-level resolution is strongly preferred for histocompatibility sequencing where recombined genes can exhibit different compatibilities than their parents. In other contexts, gene complementation can provide protection if deleterious mutations are found on only one allele of a gene. These problems are especially pronounced in immunological domains given the high levels of genetic diversity and recombination seen in regions like the Major Histocompatibility Complex. A new tool for analyzing Single Molecule, Real-Time (SMRT) Sequencing data – Long Amplicon Analysis (LAA) – can generate highly accurate, phased and full-length consensus sequences for multiple genes in a single sequencing run.


June 1, 2021  |  

Evaluation of multiplexing strategies for HLA genotyping using PacBio Sequencing technology.

Fully phased allele-level sequencing of highly polymorphic HLA genes is greatly facilitated by SMRT Sequencing technology. In the present work, we have evaluated multiple DNA barcoding strategies for multiplexing several loci from multiple individuals, using three different tagging methods. Specifically MHC class I genes HLA-A, -B, and –C were indexed via DNA Barcodes by either tailed primers or barcoded SMRTbell adapters. Eight different 16-bp barcode sequences were used in symmetric & asymmetric pairing. Eight DNA barcoded adapters in symmetric pairing were independently ligated to a pool of HLA-A, -B and –C for eight different individuals, one at a time and pooled for sequencing on a single SMRT Cell. Amplicons generated from barcoded primers were pooled upfront for library generation. Eight symmetric barcoded primers were generated for HLA class I genes. These primers facilitated multiplexing of 8 samples and also allowed generation of unique asymmetric pairings for simultaneous amplification from 28 reference genomic DNA samples. The data generated from all 3 methods was analyzed using LAA protocol in SMRT analysis V2.3. Consensus sequences generated were typed using GenDx NGS engine HLA-typing software.


June 1, 2021  |  

Assembly of complete KIR haplotypes from a diploid individual by the direct sequencing of full-length fosmids.

We show that linearizing and directly sequencing full-length fosmids simplifies the assembly problem such that it is possible to unambiguously assemble individual haplotypes for the highly repetitive 100-200 kb killer Ig-like receptor (KIR) gene loci of chromosome 19. A tiling of targeted fosmids can be used to clone extended lengths of genomic DNA, 100s of kb in length, but repeat complexity in regions of particular interest, such as the KIR locus, means that sequence assembly of pooled samples into complete haplotypes is difficult and in many cases impossible. The current maximum read length generated by SMRT Sequencing exceeds the length of a 40 kb fosmid; it is therefore possible to span an entire fosmid in one sequencing read. Shearing, sequencing and assembling fosmids in a shotgun approach is prone to errors when the underlying sequence is highly repetitive. We show that it is possible to directly sequence linearized fosmids and generate a high-quality consensus by simple alignment, removing the need for an error-prone assembly step. The high-quality sequence of complete fosmids can then be tiled into full haplotypes. We demonstrate the method on DNA samples from a number of individuals and fully recover the sequence of both haplotypes from a pool of KIR fosmids. The ability to haplotype and sequence complex immunogenetic regions will bring exciting opportunities to explore the evolution of disease associations of the immune sub-genome. This simple and robust approach can be scaled-up allowing a complex genomic region to be sequenced at a population level. We expect such sequencing to be valuable in disease association research.


June 1, 2021  |  

Full-length sequencing of HLA class I genes of more than 1000 samples provides deep insights into sequence variability

Aim: The vast majority of donor typing relies on sequencing exons 2 and 3 of HLA class I genes (HLA-A, -B, -C). With such an approach certain allele combinations do not result in the anticipated “high resolution” (G-code) typing, due to the lack of exon-phasing information. To resolve ambiguous typing results for a haplotype frequency project, we established a whole gene sequencing approach for HLA class I, facilitating also an estimation of the degree of sequence variability outside the commonly sequenced exons. Methods: Primers were developed flanking the UTR regions resulting in similar amplicon lengths of 4.2-4.4 kb. Using a 4-primer approach, secondary primers containing barcodes were combined with the gene specific primers to obtain barcoded full-gene amplicons in a single amplification step. Amplicons were pooled, purified, and ligated to SMRT bells (i.e. annealing points for sequencing primers) following standard protocols from Pacific Biosciences. Taking advantage of the SMRT chemistry, pools of 48-72 amplicons were sequenced full length and phased in single runs on a Pacific Biosciences RSII instrument. Demultiplexing was achieved using the SMRT portal. Sequence analysis was performed using NGSengine software (GenDx). Results: We successfully performed full-length gene sequencing of 1003 samples, harboring ambiguous typings of either HLA-A (n=46), HLA-B (n=304) or HLA-C (n=653). Despite the high per-read raw error rates typical for SMRT sequencing (~15%) the consensus sequence proved highly reliable. All consensus sequences for exons 2 and 3 were in full accordance with their MiSeq-derived sequences. Unambiguous allelic resolution was achieved for all samples. We observed novel intronic, exonic as well as UTR sequence variations for many of the alleles covered by our data set. This included sequences of 600 individuals with HLA-C*07:01/C*07:02 genotype revealing the extent of sequence variation outside the exons 2 and 3. Conclusion: Here we present a whole gene amplification and sequencing approach for HLA class I genes. The maturity of this approach was demonstrated by sequencing more than 1000 samples, achieving fully phased allelic sequences. Extensive sequencing of one common allele combination hints at the yet to discover diversity of the HLA system outside the commonly analyzed exons.


June 1, 2021  |  

Phased full-length SMRT Sequencing of HLA DPB1

Aim: In contrast to exon-based HLA-typing approaches, whole gene genotyping crucially depends on full-length sequences submitted to the IMGT/HLA Database. Currently, full-length sequences are provided for only 7 out of 520 HLA-DPB1 alleles. Therefore, we developed a fully phased whole-gene sequencing approach for DPB1, to facilitate further exploration of the allelic structure at this locus. Methods: Primers were developed flanking the UTR-regions of DPB1 resulting in a 12 kb amplicon. Using a 4-primer approach, secondary primers containing barcodes were combined with the gene-specific primers to obtain barcoded full-gene amplicons in a single amplification step. Amplicons were pooled, purified, and ligated to SMRT bells (i.e. annealing points for sequencing primers) following standard protocols from Pacific Biosciences. Taking advantage of the SMRT chemistry, pools of 48 amplicons were sequenced full length in single runs on a Pacific Biosciences RSII instrument. Demultiplexing was performed using the SMRT portal. Sequence analysis was performed using the NGSengine software (GenDx). Results: We analyzed a set of 48 randomly picked samples. With 3 exceptions due to PCR failure, all genotype assignments conformed to standard genotyping results based on exons 2 and 3. Allelic proportions for heterozygous positions were evenly distributed (range 0.4 – 0.6) for all samples, suggesting unbiased amplifications. Despite the high per-read raw error rates typical for SMRT sequencing (~15%) the consensus sequence proved highly reliable. All consensus sequences for exons 2 and 3 were in full accordance with their MiSeq-derived sequences. We describe novel intronic sequence variation of the 7 so far genomically defined alleles, as well as 7 whole-length DPB1 alleles with hitherto unknown intronic regions. One of these alleles (HLA-DPB1*131:01) is classified as rare. Conclusion: Here we present a whole gene amplification and sequencing workflow for DPB1 alleles utilizing single molecule real-time (SMRT) sequencing from Pacific Biosciences. Validation of consensus sequences against known exonic sequences highlights the reliability of this technology. This workflow will facilitate amending the IMGT/HLA Database for DPB1.


June 1, 2021  |  

Whole gene sequencing of KIR-3DL1 with SMRT Sequencing and the distribution of allelic variants in different ethnic groups

The killer-cell immunoglobulin-like receptor (KIR) gene family are involved in immune modulation during viral infection, autoimmune disease and in allogeneic stem cell transplantation. Most KIR gene diversity studies and their impact on the transplant outcome is performed by gene absence/presence assays. However, it is well known that KIR gene allelic variations have biological significance. Allele level typing of KIR genes has been very challenging until recently due to the homologous nature of those genes and very long intronic sequences. SMRT (Single Molecule Real-Time) Sequencing generates average long reads of 10 to 15 kb and allows us to obtain in-phase long sequence reads. We have developed a PCR assay for SMRT Sequencing on the PacBio RS II platform in our lab for 3DL1 whole gene sequencing. This approach allows us to obtain allele level typing for 3DL1 genes and could serve as a model to type other KIR genes at allelic level.


June 1, 2021  |  

Collection of major HLA allele sequences in Japanese population toward the precise NGS based HLA DNA typing at the field 4 level

We previously reported on the use of the Ion PGM next generation sequencing (NGS) platform to genotype HLA class I and class II genes by a super-high resolution, single-molecule, sequence-based typing (SS-SBT) method (Shiina et al. 2012). However, HLA alleles could not be assigned at the field 4 level at some HLA loci such as DQA1, DPA1 and DPB1 because the SNP and indel densities were too low to identify and separate both of the phases. In this regard, we have now added the single molecule, real-time (SMRT) DNA sequencer PacBio RS II method to our analysis in order to test whether it might determine the HLA allele sequences in some of the loci with which we previously had difficulties. In this study, we report on sequence-based genotyping of entire HLA gene sequences from the promoter-enhancer region to 3’UTR of the major HLA loci (A, B, C, DRB1, DRB345, DQA1, DQB1, DPA1 and DPB1) using 46 Japanese reference subjects who represented a distribution of more than 99.5% of the HLA alleles at each of the HLA loci and the PacBio RS II and Ion PGM systems.


June 1, 2021  |  

Analysis of 37,000 Caucasian samples reveals tight linkage between SNP RS9277534 and high resolution typing of HLA-DPB1

HLA-DPB1 mismatching between patients and unrelated donors is known to increase the risk of acute graft-versus-host-disease (GvHD) after hematopoietic stem cell transplantation. If only HLA-DPB1 mismatched donors are available, the genotype defined by the Single Nucleotide Polymorphism (SNP) rs9277534 can be used to select mismatched donors that are well-tolerated. However, since rs9277534 resides within the 3’ untranslated region (UTR), it usually is not analyzed during DPB1 routine typing.


June 1, 2021  |  

Characterizing haplotype diversity at the immunoglobulin heavy chain locus across human populations using novel long-read sequencing and assembly approaches

The human immunoglobulin heavy chain locus (IGH) remains among the most understudied regions of the human genome. Recent efforts have shown that haplotype diversity within IGH is elevated and exhibits population specific patterns; for example, our re-sequencing of the locus from only a single chromosome uncovered >100 Kb of novel sequence, including descriptions of six novel alleles, and four previously unmapped genes. Historically, this complex locus architecture has hindered the characterization of IGH germline single nucleotide, copy number, and structural variants (SNVs; CNVs; SVs), and as a result, there remains little known about the role of IGH polymorphisms in inter-individual antibody repertoire variability and disease. To remedy this, we are taking a multi-faceted approach to improving existing genomic resources in the human IGH region. First, from whole-genome and fosmid-based datasets, we are building the largest and most ethnically diverse set of IGH reference assemblies to date, by employing PacBio long-read sequencing combined with novel algorithms for phased haplotype assembly. In total, our effort will result in the characterization of >15 phased haplotypes from individuals of Asian, African, and European descent, to be used as a representative reference set by the genomics and immunogenetics community. Second, we are utilizing this more comprehensive sequence catalogue to inform the design and analysis of novel targeted IGH genotyping assays. Standard targeted DNA enrichment methods (e.g., exome capture) are currently optimized for the capture of only very short (100’s of bp) DNA segments. Our platform uses a modified bench protocol to pair existing capture-array technologies with the enrichment of longer fragments of DNA, enabling the use of PacBio sequencing of DNA segments up to 7 Kb. This substantial increase in contiguity disambiguates many of the complex repeated structures inherent to the locus, while yielding the base pair fidelity required to call SNVs. Together these resources will establish a stronger framework for further characterizing IGH genetic diversity and facilitate IGH genomic profiling in the clinical and research settings, which will be key to fully understanding the role of IGH germline variation in antibody repertoire development and disease.


June 1, 2021  |  

The MHC Diversity in Africa Project (MDAP) pilot – 125 African high resolution HLA types from 5 populations

The major histocompatibility complex (MHC), or human leukocyte antigen (HLA) in humans, is a highly diverse gene family with a key role in immune response to disease; and has been implicated in auto-immune disease, cancer, infectious disease susceptibility, and vaccine response. It has clinical importance in the field of solid organ and bone marrow transplantation, where donors and recipient matching of HLA types is key to transplanted organ outcomes. The Sanger based typing (SBT) methods currently used in clinical practice do not capture the full diversity across this region, and require specific reference sequences to deconvolute ambiguity in HLA types. However, reference databases are based largely on European populations, and the full extent of diversity in Africa remains poorly understood. Here, we present the first systematic characterisation of HLA diversity within Africa in the pilot phase of the MHC Diversity in Africa Project, together with an evaluation of methods to carry out scalable cost-effective, as well as reliable, typing of this region in African populations.To sample a geographically representative panel of African populations we obtained 125 samples, 25 each from the Zulu (South Africa), Igbo (Nigeria), Kalenjin (Kenya), Moroccan and Ashanti (Ghana) groups. For methods validation we included two controls from the International Histocompatibility Working Group (IHWG) collection with known typing information. Sanger typing and Illumina HiSeq X sequencing of these samples indicated potentially novel Class I and Class II alleles; however, we found poor correlation between HiSeq X sequencing and SBT for both classes. Long Range PCR and high resolution PacBio RS-II typing of 4 of these samples identified 7 novel Class II alleles, highlighting the high levels of diversity in these populations, and the need for long read sequencing approaches to characterise this comprehensively. We have now expanded this approach to the entire pilot set of 125 samples. We present these confirmed types and discuss a workflow for scaling this to 5000 individuals across Africa.The large number of new alleles identified in our pilot suggests the high level of African HLA diversity and the utility of high resolution methods. The MDAP project will provide a framework for accurate HLA typing, in addition to providing an invaluable resource for imputation in GWAS, boosting power to identify and resolve HLA disease associations.


April 21, 2020  |  

A comparison of immunoglobulin IGHV, IGHD and IGHJ genes in wild-derived and classical inbred mouse strains.

The genomes of classical inbred mouse strains include genes derived from all three major subspecies of the house mouse, Mus musculus. We recently posited that genetic diversity in the immunoglobulin heavy chain (IGH) gene loci of C57BL/6 and BALB/c mice reflect differences in subspecies origin. To investigate this hypothesis, we conducted high-throughput sequencing of IGH gene rearrangements to document IGH variable (IGHV), joining (IGHJ), and diversity (IGHD) genes in four inbred wild-derived mouse strains (CAST/EiJ, LEWES/EiJ, MSM/MsJ, and PWD/PhJ), and a single disease model strain (NOD/ShiLtJ), collectively representing genetic backgrounds of several major mouse subspecies. A total of 341 germline IGHV sequences were inferred in the wild-derived strains, including 247 not curated in the International Immunogenetics Information System. In contrast, 83/84 inferred NOD IGHV genes had previously been observed in C57BL/6 mice. Variability among the strains examined was observed for only a single IGHJ gene, involving a description of a novel allele. In contrast, unexpected variation was found in the IGHD gene loci, with four previously unreported IGHD gene sequences being documented. Very few IGHV sequences of C57BL/6 and BALB/c mice were shared with strains representing major subspecies, suggesting that their IGH loci may be complex mosaics of genes of disparate origins. This suggests a similar level of diversity is likely present in the IGH loci of other classical inbred strains. This must now be documented if we are to properly understand inter-strain variation in models of antibody-mediated disease. This article is protected by copyright. All rights reserved.This article is protected by copyright. All rights reserved.


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