hla sequencing - the barcode on your cells
DESCRIPTION
Slides that walk you through the basics of why the HLA region in the genome is important, and how to analyze it by next generation sequencing.TRANSCRIPT
HLA Typing:Understanding the
BARCODE on your cells.
Swati Kadam, Ph.D.
Barcodes help identify cells
What is HLA?• HLA = Human Leukocyte Antigen• Immune cells read HLA-barcode on cells to help identify
self vs. non self cells or uninfected vs. infected cells.– An immune response is triggered if a infected cell is identified.
Y
Y YY
Y Y
Y Y
Y
Y YYY Y
Y Y
Y
Y YY
Y Y
Y Y
Y
Y YY
Y Y
Y Y
Infected cell
T-cell
Uninfected cell
Uninfected cell
Uninfected cell
• Any cell displaying some other HLA type is "non-self" and is seen as an invader by the body's immune system, resulting in the rejection of the tissue bearing those cells.
Y
Y YY
Y Y
Y Y
Y
Y YYY Y
Y Y
Y
Y YY
Y Y
Y Y
YY YY
Y Y
Y Y
Non-self cell : INVADER
T-cell
Self cell
Self cell
Self cell
What is HLA?
MHC Region
MHC Class II genes MHC Class III genes MHC Class I genes
HLA-AHLA-C
TNF C4
HLA-DQHLA-DP HLA-DR
HLA-B
MHC houses HLA genesChromosome 6
MHC Region
MHC Class II genes MHC Class III genes MHC Class I genes
HLA disease associationsChromosome 6
- Leprosy- Multiple Sclerosis- Lymphoid Leukemia- Rh(D) isoimmunization- Psoriasis- Ankylosing spondylitis- Hemophilia with synovitis- Malaria- Susceptibility or Resistance to HIV-1- Type1 autoimmune hepatitis- ANCA-positive autoimmune disease
- Multiple Sclerosis- Psoriasis- Systemic Lupus- Asthma- Childhood Acute Lymphoblastic Leukemia (ALL)- HIV-related disease- Thyroid Carcinoma- Nephropathy- Kawasaki disease- Celiac Disease
http://ccg.murdoch.edu.au/private/yurek_kulski/mhc/Table4.html
HLA disease associations
HLA status and control over HIV infection
- HIV controllers = HIV positive individuals that do not develop a clinical disease and do not progress to AIDS.
- HLA Class I SNPs can determine whether you are a HIV controller or progressor http://aids.gov/hiv-aids-basics/hiv-aids-101/what-is-hiv-aids/
X
X
HLA drug hypersensitivity testing
Allergy to Abacavir is strongly associated with the presence of the HLA-B*5701 gene
Gluten intolerance
• Celiac disease is an autoimmune disorder of the small intestine triggered by gluten consumption.
• Over 95% of people with celiac have the isoform of DQ2 or DQ8, on Class II MHC genes.
• Organ transplantation• Hematopoietic stem cell transplant• HLA types associated with specific immune
diseases.• Parental testing• Reduce instance of GVHD– Non-related donor– Related donor
Importance of HLA typing
Did you know?• In the United States:
– Nearly 120,000 need lifesaving organ transplants.
– Every 10 minutes another name is added to the national organ transplant waiting list.
– An average of 6000 people die each year from the lack of available organs for transplant.
– In 2012, there were 14,013 Organ Donors resulting in 28,052 organ transplants.
– More than 1 million tissue transplants are done each year and the surgical need for tissue has been steadily rising.
• $230M worldwide transplant test market.
1, 8, 103, 14, 17
10, 16, 82, 7, 11
1, 8, 10 1, 8, 102, 7, 11 10, 16, 8
3, 14, 172, 7, 11
3, 14, 1710, 16, 8
Mother Father
Daughter 1 Daughter 2Son 1 Son 2
10, 16, 8
Son 3
1, 8, 10
Variation in HLA alleles even between siblings.Random and varied distribution of Haplotypes from each parent.
High resolution typing provides a more accurate match
Finding a match is not easy!
Match
Savior Siblings
• Serotyping– Detection of antibodies is serum (non
sequencing based method).
• Sanger sequencing of amplified regions– Amplicon sequencing of specific exon regions.
• Sequence Specific Oligonucleotide Hybridization (SSO)– Oligo hybridization based detection of allele
status.
• (NGS) MHC sequencing for donor matching– Sequencing of entire MHC region.– Sequencing of amplicons for target regions.
Typing Methods
Method About method Pros Cons
SerotypingNon-sequencing based typing method where antibodies specific to HLA proteins are used to identify the proteins on the cell surface.
- Low Cost- Rapid- Traditional
- Crude Method- Protein based detection- Inaccurate typing- Protein binding to more
than one serotype
Sequence Specific Oligonucleotide
Hybridization (SSO)
Typing method where specific oligos are first designed for genes of interest and then hybridized to patient or donor DNA to check for hybridization.
- Checking of specific target
- Efficient
- Cannot account for unrecorded alleles
- Hybridization errors- Need to know target
sequence- Cannot phase
Sanger SequencingSanger sequencing or Sequencing by Termination (SBT) is a classical method used for sequencing specific regions of the MHC.
- Used to sequence regions of interest
- Fast- Base pair resolution- Coverage only 2x
- Different HLA alleles share similar sequences, difficulty aligning.
- Cannot phase
Next-Gen SeqPerforming long range PCR to amplify HLA genes in MHC region, fragmenting the amplified genes and then preforming deep sequencing.
- Deep coverage (1000x)- Total MHC coverage- Rapid high throughput- Accurate and efficient- Phasing
- Data Analysis
Typing Methods
HLA nomenclature
http://hla.alleles.org/
We have just scratched the surface
Table S1: PCR primers and estimated amplicon size for the 6 HLA genes
Locus Forward primer Seq Reverse primer Seq Length (bp)
HLA-A HLA-A_F7 ATCCTGGATACTCACGACGCGGAC HLA-A_R8 CATCAACCTCTCATGGCAAGAATTT 3398
HLA-C HLA-C_F4 GGCCGCCTGTACTTTTCTCAGCAG HLA-C_R3 CCATGGTGAGTTTCCCTGTACAAGAG 4440HLA-B HLA-B_F3 AGGTGAATGGCTCTGAAAATTTGTCTC HLA-B_R3 AGAGTTTAATTGTAATGCTGTTTTGACACA 4296HLA-DRB1 HLA-DRB1_F1 TGATTGACTTGCTGGCTGGTTTCTCATC HLA-DRB1_R2 GCATCCACAGAATCACATTTTCTAGTGTT 11899HLA-DQB1 HLA-DQB1_F6 TCATGTGCTTCTCTTGAGCAGTCTGA HLA-DQB1_R7 TGTGACAGCAATTTTCTCTCCCCT 7118HLA-DPB1 HLA-DPB1_F1 TGGTCCAACAGGATCACATTTATAAGTGT HLA-DPB1_R1 CCCAGTTTGGATGGTCTCTCAGCTCTT 13605
13,605bp 7,118bp 11,899bp
4,296bp 4,440bp 3,398bp
Long range PCR
Nextera
MiSeq
• Nextera/MiSeq– Coverage of coding and non-coding HLA
regions.– Nextera tagmentation protocol provides easier
faster processing of samples– Greater depth delivers accuracy exceeding
existing technologies.
Sequencing
1, 8, 103, 14, 17
10, 16, 82, 7, 11
1, 8, 10 1, 8, 102, 7, 11 10, 16, 8
3, 14, 172, 7, 11
3, 14, 1710, 16, 8
Mother Father
Daughter 1 Daughter 2Son 1 Son 2
10, 16, 8
Son 3
1, 8, 10
Variation in HLA alleles even between siblings.Random and varied distribution of Haplotypes from each parent.
Finding a match is not an easy task.
Unique HLA fingerprint
Match
• MiSeq install at HistoGenetics and DKMS Life Science Lab in Germany.
High resolution HLA typing because: – Read length (2x250, 2x300) allows phasing– Highest accuracy and speed– 1000x Coverage
• (consensus accuracy 99.999%)
– No Homo polymer errors– Easier workflow
MiSeq leads the way
• Analyze SNPs, allelic differences, status of microsatellites, and HLA typing all in one.
• Basic SNP or genotyping analysis tool
• IMGT-HLA database
Data Analysis
LET’S START DECIPHERING THE HLA-BARCODE.
Questions : Swati kadam, Ph.D. [email protected]
Questions : Abizar [email protected]
Appendix
• Currently at about 50 HLA related MiSeq installs in the last few months– DKMS. From Martin Allgaier’s install report: “DKMS Life Science Lab, a subsidiary of
DKMS, is a state-of-the-art HLA tissue typing laboratory in Dresden, Germany performing all sequencing activities to MiSeq. This was the 11th (!!!) Miseq for this customer and they are still planning for more. Currently, there are no signs of switching to another platform as the MiSeq gives them the output and flexibility they need for their service.”
– HistoGenetics. From Peter Kraus’ install report: “MiSeq Install Report for the first 10 of 20 total MiSeqs to be installed at HistoGenetics in Ossining, NY. HistoGenetics is a company that performs HLA typing as a service using sequencing assays” • Press release."We anticipate building on our leadership position and established
success by leveraging the MiSeq platform," said Dr. Nezih Cereb, Chief Executive Officer and Co-founder of HistoGenetics. "Based on our experience with other technologies, we think the MiSeq's quality data output and simple workflow, combined with Illumina's commitment to work collaboratively, is the ideal solution to enable us to provide our customers with the superior results and turnaround time they require."
Example: MiSeq installs in HLA labs
• Research customers– Association of HLA variants
with disease• Inflammatory, infection
susceptibility, cancer, etc.• More accurate cataloging of
HLA types
• Translational customers• Tissue registries• Marker validation for diseases
• Clinical customers• Transplantation match
– Registry– Confirmatory
HLA sequencing: Business Opportunity
$230M worldwide
Software companies
Conexio • SW market leader. Sanger legacy
Omixon • FDA-compliance
GenDx • mature SW, nice UI