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«АКТУАЛЬНЫЕ ВОПРОСЫ ИММУНОГЕНЕТИКИ И ТКАНЕВОГО ТИПИРОВАНИЯ»
Санкт-Петербург 24-25 июня 2015 г.
out our historical expanse. Then maternally inherited genetic traits will be explored by mitochondrial DNA analysis to ensure that a maternal counterpart
to the paternal line of descent will be available as a necessary complement to present genetic background of the Armenians.
Naumova E.
Department of Clinical Immunology and Stem Cell Bank, University Hospital «Alexandrovska», Medical University, Sofia, Bulgaria
STRATEGY FOR FINDING THE SUITABLE DONOR IN HSCT
Haematopoietic stem cell transplantation (HSCT) is limited by finding a suitable donor. The »best» donor is HLA matched sibling or unrelated donor. Unfortunately, in most cases the probability to find HLA identical sibling does not exceed 30 % and depends on the family size. Additionally, number of factors restrict the probability to find HLA matched donor in Bone Marrow Donors Worldwide such as patient's ethnicity, presence of rare alleles or haplotypes, possible relevance of «non-classical» HLA and non-HLA alleles. All these factors should be considered for the estimation of time for donor search. For the Bulgarian patients the mean time to identify 10/10 matched donor varies from 28 to more than 90 days depending on the HLA genotype. Patient's related factors such as diagnosis and disease stage and transplant protocol are also important
for donor selection and clinical outcome of HCST. In order to increase the efficacy of transplantation alternative strategies such as umbilical cord blood, mismatched unrelated adult donor or haploidenti-cal related donor are considered for patients lacking HLA identical donor. In such cases it is important to estimate a number of clinically relevant factors, such as: the time for engraftment and haematopoie-sis reconstitution, graft failure rate, graft versus host disease, transplant related mortality and relapses. Current data in the literature as well as our experience on the relevance of immunogenetic factors in HSCT will be discussed. An algorithm for finding the most suitable donor, that could help clinicians to provide an adequate treatment for each individual patient will be presented.
Nezih Cereb, MD
CEO & Co-founder Histogenetics
ADVANCES IN DNA SEQUENCING TECHNOLOGIES FOR HIGH RESOLUTION HLA TYPING
Recent advances in DNA sequencing technologies, so-called Next Generation Sequencing (NGS), have brought breakthroughs in deciphering the genetic information in all living species at a large scale and at an affordable level.
By introducing DNA barcode (index) sequences multiplexing samples from hundreds of individual became possible for genotyping certain genomic regions faster and cheaper with higher resolution.
In this talk I will present Histogenetics's experience and accomplishments in applying NGS for large-scale high resolution HLA typing. Histogenet-
ics had established Sanger capillary technology in 2006 for large volume DNA-based sequencing typing and more than 3.8 million samples were typed with that technique. Histogenetics' existing infrastructure helped us to transition to the NGS technologies without compromising accuracy, volume of typing and speed. In March 2013 Histogenetics introduced a Hybrid approach of Sanger + Illumina MiSeq DNA sequencing. A total 460,190 samples were typed with MiSeq + Sanger to validate MiSeq data during transition to NGS, shown in the table 1.
BECTHHKiEMATOnOiHH, tomXI, №2, 2015
Table 1.
Total A B C DRB1 DRB3 DRB4 DRB5 DQB1 DQA1 DPB1 DPA1
460,190 404,016 410,523 393,343 424,154 178,933 159,060 180,648 325,989 11,775 268,896 9,679
High resolution typing was achieved using NGS MisSeq platform. Comparison of resolution level
After establishing the new platform, in October 2013 we introduced Illumina MiSeq as the first line method for high volume, high resolution HLA Typing. To date we have typed close to 5 million individuals using SBT. While we were pushing for higher volume typing, we were also exceling in quality and accuracy with the strict quality control and quality assurance policy es-
In spite of these excellent results with Illumina MiSeq technology we have been exploring other single molecule sequencing technologies such as Pacific Bioscience's RS II.
The MiSeq platform has accomplished higher resolution HLA typing results, faster and more cost effective and easier work flow compared to Sanger Sequencing and other NGS. However, it has some shortcomings such as shorter read length compared to Sanger and PacBio that could result in missing insertions, and inability in phasing the exons unless additional amplicons are introduced. In addition MiSeq has a long run time and produces sequencing artifact in certain amplicons. Also, depending on a single technology and company can be a risk when quality of reagents fails or becomes substandard.
The PacBio platform has the following advantages to the MiSeq platform: Long read lengths with excellent phasing of the Exons and Introns and
between NGS and Sanger sequencing techniques for registry donors are shown below (Table 2).
Table 2.
tablished in Histogenetics' High throughput HLA typing process.
National Marrow Donor Program (NMDP) is one of the Histogenetics' major clients, and has a strict quality control program where average 3 % of blind QC samples are included in every batch of testing samples. The table 3 shows error free typing for NMDP registry donors.
Table 3.
short run times. It also provides us with an excellent alternative technology. Disadvantages of PacBio compared to MiSeq are a limitation in the barcoding (multiplexing) and longer sample preparation time.
Since October 2014, we have been routinely using PacBio for class I typing for resolving exon shuffling ambiguities and the new alleles. We have performed more than 5000 HLA-ABC on the PacBio platform, sequencing 1 kb amplicon that include ARS region (exon 2 and exon 3). We are incrementally extending the coverage length, and now for special projects we can routinely type the full gene length -3.5 kb which includes 8 exons and seven introns. Typing full length Class II genes are more challenging due to the lengths. They are approximately 18 kb or longer. But typing 5 kb fragments that include exon 2 and the rest of the downstream exons to the 3' translated regions are underway.
LOCUS HIGH Resolution result percentage
NGS Sanger
HLA-A 99.94 75.6
HLA-B 99.75 80
HLA-C 97.75 60
HLA-DRB1 100.00 95.5
HLA-DQB1 100.00 96.1
HLA-DPB1 100.00 98
Total (years: 2011-2015)
Samples Typed 716,540
Blind QC Samples 18,714
QC Error 0
«АКТУАЛЬНЫЕ ВОПРОСЫ ИММУНОГЕНЕТИКИ И ТКАНЕВОГО ТИПИРОВАНИЯ»
Санкт-Петербург 24-25 июня 2015 г.
Another very important issue with NGS is the interpretation, presentation and visualization of the data. The focus should be matching patients and potential donors for those regions defining Antigen Recognition Sites (ARS) unambiguously-while not-
ing the similarities and variations in other regions of the gene.
Below (Figure 1) is an example of presentation for sequence matching at ARS between patient and potential donors.
Figure 1.
Genes H LA-A HIA-B HIA-C HLA-DRB1 HLA-DQB1
Exons El ARS(E2+E3] E4 El ARS (E2+EÎ) E4 El ARS(E2+EJ) E4 E7 ARS (E2] E3 ARS (E2) E3
Alleles by ARS 01:01:016 15:02:016 04:01:016 13:01:016 05:03:016
Patient 68:01:026 51:01:016 07:01:016 14:01:016 06:03:016
E1.0005 ARS. 0001 E4.0004 E1.0012 ARS.0004 E4.0015 E1.0023 ARS.0004 E4.0003 E7.0001 ARS.0013 E3.0005 ARS.0005 E3.0020
E1.0004 ARS. 0015 E4.0004 E1.0013 ARS.0005 E4.0100 E1.0030 ARS.0007 E4.0018 E7.0002 ARS.0014 E3.0007 ARS.0006 E3.0020
10 / i 0 match то the Patient (ARS)
Donor 1 Alleles by ARS 01:01:016 15:02:016 04:01:016 13:01:016 05:03:016
68:01:026 51:01:016 07:01:016 14:01:016 06:03:016
1 E1.0005 ARS. 0001 E4.0004 E1.0012 ARS.0004 E4.0015 E1.0023 ARS.0004 E4.0003 E7.0001 ARS.0013 E3.0005 ARS.0005 E3.0020
| El.0004 ARS.0015 E4.0004 E1.0013 ARS.0005 E4.0100 E1.0030 ARS.0007 E4.0018 E7.0002 ARS.0014 E3.0007 ARS.0006 E3.0020
10/10 Hatch to the Patient (ARS)
Donor 2 Alleles by ARS 01:01:016 15:02:016 04:01:016 13:01:016 05:03:016
68:01:026 51:01:016 07:01:016 14:01:016 06:03:016
1 E1.0005 ARS. 0001 E4.0001 E1.0012 ARS.0004 E4.0017 El,0024 ARS.0004 E4.0003 E7.0003 ARS.0013 E3.0005 ARS.0005 E3.Ü021
Щ El.0005 ARS.0015 E4.0004 El.0014 ARS.0005 E4.0101 El.0031 ARS.0007 E4.0001 E7.0002 ARS.0014 E3.00C1 ARS.0006 E3.0021
5 / i 0 Match to the Patient (ARS)
Donor 3 Alleles by ARS 01:01:016 07:05:016 04:02:016 13:01:016 05:03:016
24:02:016 51:01:016 18:01:016 15:01:016 06:02:016
Indexl ARS. 0001 ARS.0006 ARS.0004 ARS.0013 ARS.0005
IndexZ ARS.0016 ARS.0005 ARS.0018 ARS.0015 ARS.0006
*ARS: Antigen Recognition Site.
* G Code: G code is a group of alleles that have identical nucleotide sequences in the antigen recognition site
Above figure is a schematic presentation of HLA typing report that compares patient and potential donors focusing on ARS regions.
Conclusion. Recent progress in sequencing technologies and laboratory processes together with
advanced informatics enable us to have a clearer representation of MHC and other Immune response genes. This will in turn help us to understand the puzzles of complex genetic systems that can serve the base for health and disease.
