HLA-E genotyping – the sooner the better
J. Diegel (Birkenfeld, DE)
The Human Leukocyte Antigen E belongs to the non-classical HLA class I molecules located within the MHC on chromosome 6p21 is and described to display only limited polymorphism. The HLA-E complex is the major ligand for the natural killer inhibitory receptor CD94/NKG2A and the activating receptor CD94/NKG2C. The complex can also present peptides originating from the cellular antigen processing pathway and therefore induce T-cell mediated immune responses. Nowadays there is growing evidence that HLA-E polymorphisms have a major impact on the immune system and most likely affect hematopoietic stem cell transplantation outcome. To evaluate HLA-E polymorphism and their possible impact on transplantation we designed a NGS based long range PCR assay covering the entire gene. Due to the lack of suitable reference samples we sequenced 95 randomly selected DNA samples to prove amplification of so far described HLA-E alleles. The generated NGS data were analyzed with the HLA Twin software (Omixon) and confirmed by Sanger sequencing using a freeware tool (BioEdit). During analysis we found several so far undescribed sequence variants mainly located in the non-coding sections. One sample revealed a nucleotide substitution from G to A at position 1644 in exon 4 responsible for an amino acid change in codon 196 from Asp to Arg.From this observation we conclude that the frequency of polymorphism is higher than so far estimated. Implementing this in-house designed, full-length HLA-E sequencing assay into our NGS HLA typing routine will probably lead to the detection of more variants increasing our knowledge about HLA-E, hopefully to the benefit of transplantation outcome
ABO blood group typing with Oxford Nanopore MinION sequencing
B. Matern (Maastricht, NL, NL)
Typing of the ABO blood groups, which encodes the transferase that catalyzes the last step in the synthesis of the A, B, and H antigens, is a prerequisite for both the recipient and donor in solid organ transplantation, stem cell transplantation, and blood transfusion. Historically, blood group typing has typically been done with serology, but in a number of cases, such as registry typing with buccal swabs and samples from cord blood, only DNA is available. The blood group types A, B, and O show clear differences in the genetic sequence of the ABO gene, and therefore DNA sequencing offers a valuable alternative to serology in these cases. In this study we have used MinION Nanopore sequencing to obtain full length sequences of the ABO gene. Two long-range overlapping amplicons were prepared which span the complete 18kb length of the ABO gene for a set of samples which represent the six possible ABO combinations (AA, BB, AO, BO, AB and OO). The amplicons were sequenced on the Oxford Nanopore MinION, and reads were aligned with known reference sequences obtained from the NCBI dbRBC database. DNA sequence data was sufficient to differentiate between the six possible ABO combinations based on 3 polymorphic positions. Although the current analysis focuses on the polymorphism that defines the blood group types as a proof of principle, the acquired full-length sequences can provide information about the extensiveness of polymorphism present in the ABO alleles, and help complete the full-length ABO allele database. Overall, this study shows that Nanopore sequencing on the MinION represents a novel platform for full-length high-throughput sequencing of ABO genes, suitable for cases where only DNA is available.
Towards full-length sequencing: comparison of different sequencing methods
S. Montanič (Ljubljana, SI)
Excluding null alleles and resolving ambiguities within G groups are mandatory in the laboratories serving unrelated HSCT programs. Since routine sequencing strategies have mainly focused on exons 2 and 3, for more than 90% of the HLA alleles valuable full-length sequence information is lacking. The 17th IHIWG programme includes working groups for full length HLA gene sequencing. Using the full-length hemizygous Sanger sequencing method (SSBT 17th workshop protocol) we extended the sequence of a new allele, B*27:30 found in a family of Slovenian origin. In 6 individuals within family we confirmed the A*02:01-B*27:05-DRB1*01:01 haplotype, which is common in the Slovenian population. Oxford Nanopore MinION® sequencing platforms, a small and low-cost single-molecule sequencer, which offers the possibility of sequencing long DNA fragments, was used for retyping all samples as well as for an additional nine randomly chosen individuals previously typed by Sanger SBT (AlleleSEQR HLA-SBT Reagents, Abbott) and another NGS platform (Roche reagents with the Roche GS Junior system). We obtained complete HLA-A, -B and -C full length sequences of all analyzed samples with SSBT that provide reference data for NGS. Raw data produced by MinION were concordant with SSBT as well as with EFI standards requirements enabling allelic resolution typing result interpreted by adequate commercial software. In conclusion, we find SBT with Oxford Nanopore MinION® device as very promising method to be used both in high throughput, but definitely also in medium to small size H&I laboratories.
Isolation of very pure lymphoid (CD3+, CD19+ or CD56+) and myeloid (CD15+ or CD33/CD66b+) cell subsets in as little as 15 minutes for use in lineage-specific chimerism analysis
K. McQueen (Vancouver, CA)
Chimerism analysis is used to monitor the presence of donor leukocytes in a recipient following haematopoietic cell transplantation. Lineage-specific chimerism can increase sensitivity compared to analyzing the entire leukocyte population, however it requires the isolation of highly purified cell subsets, as even a few contaminating cells can compromise the integrity of the assay. We describe a method (EasySep) to rapidly isolate very pure lymphoid (CD3+, CD19+ or CD56+) and myeloid (CD15+ or CD33/CD66b+) cell subsets directly from whole blood (WB) or buffy coat (BC) in as little as 15 minutes. Briefly, blood or BC was diluted with an equal volume of a red blood cell lysis reagent and target desired cells were immunomagnetically labelled and then placed in a magnet. Target cells were retained in the magnet while unwanted cells were poured or pipetted off. Target cell purities obtained after separation were as follows (mean +/- SD): CD3+: 99% +/-1 (n=6); CD19+: 98% +/-1 (n=4); CD56+: 99% +/-1 (n=4); CD15+: 98% +/-1 (n=10); myeloid (CD33+ and CD66b+): 99% +/-2 (n=18). Starting from 1 mL of WB, 900,000 CD3+ T cells, 120,000 CD19+ B cells, 2.6 million CD15+ cells or 3 million myeloid cells were obtained. Approximately 600,000 CD56+ cells were obtained from 1 mL of BC (average start number 1.4 x10e7 cells). To save time and increase laboratory throughput, EasySep immunomagnetic cell separation can be fully automated with the RoboSep instrument. The separation procedure is compatible with downstream DNA isolation methods. These new EasySep kits provide immunogenetics laboratories a fast and easy method to obtain highly purified cells for use in lineage-specific chimerism testing.
Evaluation of a commercially available HLA typing kit for NGS
A. Dinou (Athens, GR)
The HLA typing laboratory of the Hellenic Cord Blood Bank is routinely using NGS-based typing for the CBUs of its inventory at 7 loci (HLA-A, -B, -C,-DRB1, -DQA1, -DQB1 and -DPB1) on the Miseq platform, using the Holotype kit and HLA Twin software for analysis (Omixon). The aim of this study was the evaluation of the TruSight HLA v2 Sequencing Panel (Illumina) and the assign 2.0 software on 24 samples, previously typed with our validated method. The samples were processed in two batches of 12 and typed for 11 loci (HLA-A, -B, -C, -DRB1, -DRB3,4,5, -DQA1, -DQB1, -DPA1 and -DPB1). The workflow has certain differences to our routine as most steps are bead-based for the TruSight kit and do not require the use of dedicated instruments as in the Holotype kit. The total time for the 2 approaches from genomic DNA preparation to loading the Miseq are similar, as well as the hands-on time. The TruSight is low throughput, as it is only available at a 24 sample configuration and only 12 samples can be processed simultaneously. Holotype can accommodate up to 96 samples that can be processed simultaneously although for bigger number of samples automation would be desirable. Of the loci tested only one failed to yield a result (DPA1), while the results for the 7 loci tested by both methods were concordant with the exception of 4 loci where possible novelties were detected. The only ambiguities observed with TruSight were in DPB1 (due to the lack of phasing between exons 2 and 3) and in B*39:01:01:03/39:01:01:02L (due to a polymorphism in an off-target sequence). On the other hand, this kit resolves the A*02:01:01:01/02:01:01:02L ambiguity observed with the Holotype kits. The analysis software Assign 2.0 provided, gives the correct assignments but in the version tested quality indicators like fragment size, minimum depth of coverage were not easily obtainable. The Trusight HLA v2, is an easy to use kit that gives high quality typings and would be well suited for a low thoughput HLA typing laboratory.
iC-HLA cassette: An automated and integrated solution for NGS based HLA-typing
J. Han (Huntsville, US)
Based on the arm-PCR core technology and iCubate system, we have developed an easy-to-use product that allows researchers to generate sequencing-ready libraries for HLA typing automatically. The user only need load genomic DNA sample into the cassette, amplification and purification can be executed in an automated fashion by the instrument in 7 hours. The amplified library is directly ready for sequencing using the Illumina MiSeq platform. The iC-HLA cassette provides the end-user a DNA library for HLA-NGS typing in an automated, disposable and fully closed format, without complex manipulations. Compared to current HLA-NGS products, the iC-HLA cassette has some significant advantages: (1) Amplification is robust. The assay uses our patented multiplex amplification strategy, arm-PCR (amplicon rescued multiplex PCR, Patent No. 7,999,092), allowing simplified library preparation while maintaining true high end-point resolution. (2) The system is closed. Once the cassette is loaded with a sample and closed, all the amplification and purification procedures can be conducted within the cassette. Since amplicons will not be exposed to the laboratory environment, and the majority of PCR reagents are pre-installed at the manufacturing site, both the contamination source and the opportunity for contamination are under control. (3) The system is easy to use. The integrated, fully automated design minimizes requirements for specialized training and facilities for technicians who perform the test. (4) The procedure is fast. The entire NGS workflow from gDNA amplification to genotype assignment could be completed within 2 working days. With the advantages of saving time and simplifying the protocol, iC- HLA cassettes will allow laboratories to have more time and energy to analyze and interpret the results.
Complete human leukocyte antigen gene sequence determination combining long range polymerase chain reaction and next generation sequencing
T. Binder (Hamburg, DE)
Complete genomic sequence data for HLA class I and II alleles are still limited. It was our intention to develop a workflow based on long range PCR (LR-PCR) and next generation sequencing (NGS) on a MiSeq platform (Illumina) to provide the complete genomic sequence of different HLA alleles. Therefore, we designed different HLA locus and/or allele specific LR-PCRs. After amplicon generation by PCR and NGS data evaluation with two different HLA software tools (NGSengine, GenDx and Omixon Twin, Omixon) we ended up with allele-specific contigs based on phased sequence alignment according to the individual single nucleotide variants (SNVs) pattern present. The final alignment of these contigs was performed with the BioEdit (ClustalW) and AliView (Muscle) software along with published genomic sequences (IPD-IMGT/HLA Database, http://www.ebi.ac.uk/imgt/hla/). With our approach, applying LR-PCR and NGS including phased sequence analysis, we were able to determine the complete gene sequence of different HLA class I and class II alleles (from 5'-UTR to 3'UTR), separately. We are sure, that in the near future it will become much easier to identify the complete gene sequences of all HLA alleles with the combination of these methods. Apart from compatibility testing this would be helpful especially for evolutionary and ancestry studies.
2nd field HLA Typing by NGS for clinical services
M. Ahci (Essen, DE)
Although high-resolution HLA typing by Next Generation Sequencing (NGS) has entered the field several years ago, its feasibility for clinical services is still a matter of debate. Here we report on 2nd field HLA-A, -B, -DRB1, and -DPB1 typing by an in-house NGS typing system. 22 HLA-typed control B- lymphoblastoid cell lines (B-LCL) and 334 samples from patients and their unrelated, 9-10/10 HLA-matched stem cell donors were included. Typing was performed for exons 2 and 3 on an Illumina platform, according to Lange et al. (2013), with an average read-depth of 780 reads per base and bioinformatic evaluation by the neXtype software. The frequency of partial or complete drop-outs was 2%. Concordance with the reported typings for the B-LCL control cell lines was 100% for all 4 loci. NGS was able to assign unequivocal G-group or 2nd field typing in 1265/1336 (94.7%) alleles. Total turn-around time for 167 pairs without NGS-robotics was 7 days, with an average cost per sample for the 4 loci of <10€. These data demonstrate the feasibility, reliability and cost-efficiency of in-house NGS protocols for unambiguous 2nd field HLA typing for clinical services, although the use of robotics for library preparation is recommended to reduce error risks and turn-around times.