Presentation is loading. Please wait.

Presentation is loading. Please wait.

Welcome to UW-Madison, the WNPRC, and O’Connor Lab! MHC Genotyping Workshop November 7 th – 11 th, 2011 Madison, Wisconsin.

Published byAlexis Roberts Modified over 9 years ago

Similar presentations

Presentation on theme: "Welcome to UW-Madison, the WNPRC, and O’Connor Lab! MHC Genotyping Workshop November 7 th – 11 th, 2011 Madison, Wisconsin."— Presentation transcript:

1 Welcome to UW-Madison, the WNPRC, and O’Connor Lab! MHC Genotyping Workshop November 7 th – 11 th, 2011 Madison, Wisconsin

2 Introductions Trainers (WNPRC Genetics Service) – Roger Wiseman – Julie Karl – Simon Lank – Gabe Starrett – Francesca Norante Participants – Wendy Garnica – Mark Garthwaite – Julie Holister-Smith – Suzanne Queen – Premeela Rajakumar – Yuko Yuki

3 Schedule of Events Monday – Welcome and Overview Presentation – Begin bench work: cDNA synthesis & PCR (run #1) Tuesday – PCR product purification, quantification & pooling (run #1) – Begin emulsion PCR (run #1) – Begin bench work (run #2) Wednesday – Break & enrich DNA beads (run #1) – Run Roche/454 GS Junior instrument (run #1) – emPCR (run #2) Thursday – View run #1 results – Continue work on run #2 – Informatics presentation – Data analysis Friday – Run #2 results – Continue Data Analysis & Wrap-up

4 Overview of Presentation Our lab & research focus Evolution of DNA sequencing technology Discussion of Roche/454 technology & sample multiplexing MHC genotyping method overview – NHP immunogenetics – Genotyping strategy – Workflow Genotyping results

5 Welcome to Madison! WNPRC

6 Welcome to Madison!

7 The Wisconsin National Primate Research Center (WNPRC) Only federally funded National Primate Research Center in the Midwest Center holds ~1,100 rhesus macaques, 200 marmosets, and 100 cynomolgus macaques Research strengths: – Immunogenetics & Virology – Aging & Metabolism – Reproductive & Regenerative Medicine

8 The O’Connor Laboratory Genetics Services Members

9 The O’Connor Laboratory Genetics Services Members

10 The O’Connor Laboratory: Research NHP immunogenetics (MHC class I, class II, KIR) – Cynomolgus Macaque (Mauritian, Indonesian, SE Asian) – Rhesus Macaque (Indian & Chinese) – Japanese Macaque, Vervet, Sooty Mangaby SIV pathogenesis (immunology) and viral evolution Human immunogenetics (HLA) and HIV variation

11 The O’Connor Laboratory: Research NHP immunogenetics (MHC class I, class II, KIR) – Cynomolgus Macaque (Mauritian, Indonesian, SE Asian) – Rhesus Macaque (Indian & Chinese) – Japanese Macaque, Vervet, Sooty Mangaby SIV pathogenesis (immunology) and viral evolution Human immunogenetics (HLA) and HIV variation

12 Sequencing Technology is Changing Micro sequencing reactions – Pyrosequencing – Single molecule sequencing Higher throughput – Millions of sequences per day Lower cost – $10,000 human genome (original HGP = $3 billion)

13 Sequencing Technology: Overview 1 st Generation (previous): Sanger sequencing Applied Biosystems 3730xl: 1 x 10 3 reads / day - 500 to 1,000 bp read length

14 Sequencing Technology: Overview 2 nd Generation (current): 454, Illumina, SoLID, Ion torrent Roche / 454: 1 x 10 6 reads / day - 500 to 800 bp read length Illumina: 2 x 10 9 reads / week - 100 or 200 bp read length

15 Sequencing Technology: Overview 3 rd Generation (future): Pacific Biosciences, Nanopore sequencing, Complete Genomics Pacific Biosciences: 1 x 10 5 sequences / hour - 1,000 to 10,000 bp reads (?) - Single molecule sequencing - Goal = $1,000 genome !

16 Sequencing Technology: Overview 1 st Generation (previous): Sanger – Slow, Expensive, Not clonal, easy to analyze 2 nd Generation (current): 454, Illumina, SoLID, Ion torrent – Faster, Cheaper, Clonal, hard to analyze 3 rd Generation (future): Pacific Biosciences, Nanopore sequencing, Complete Genomics, Helicos – Very fast, Very cheap, Impossible to analyze

17 Roche / 454 Sequencing How does it work?

18

19

20

21

22

23 Flowgram (instead of chromat)

24 O’Connor Laboratory Sequencing 200720062008200920102005 Sanger sequencing NHP MHC class I genotyping with E. coli based cloning and Sanger sequencing: Throughput of ~ 8 animals per week.

25 O’Connor Laboratory Sequencing 200720062008200920102005 Sanger sequencing Pilot with Roche sequencing center MHC class I genotyping pilot project: ~24 samples per week

26 O’Connor Laboratory Sequencing 200720062008200920102005 Sanger sequencing GS FLX at UIUC Pilot with Roche sequencing center MHC class I genotyping at UIUC, ~ 48 samples per week

27 O’Connor Laboratory Sequencing 200720062008200920102005 Sanger sequencing GS FLX at UIUC Pilot with Roche sequencing center Titanium pilot with Roche sequencing center MHC class I full-length sequencing project with Roche using Titanium chemistry

28 O’Connor Laboratory Sequencing 200720062008200920102005 Sanger sequencing GS Junior in lab GS FLX at UIUC Pilot with Roche sequencing center Titanium pilot with Roche sequencing center MHC class I and viral sequencing projects run in- house ( > 48 samples per week )

29 Roche/454 Sequencing Advantages Inherently clonal (no bacterial cloning needed) Far cheaper per base than Sanger (3 – 4 orders of magnitude) Reliable read number and data regularity Easy protocol: many people trained

30 GS Junior 5 Month Run Summary MHC Class I 568bp Amplicon – 9 runs Average70,848 HQ reads523 bp median length Highest101,711526 Lowest33,552521 SIV Whole Genome – 16 runs Average101,846 HQ reads360 bp median length Highest177,642494 Lowest42,949147 SIV Epitope Amplicons (Various Sizes) – 5 runs Average80,244 HQ reads369 bp median length Highest107,605388 Lowest37,066356

31 Ease of Use  Access to instrument since Jan 2010  34 different fully-trained operators to date  7 additional people have begun training, but have not yet completed a solo run

32 Ease of Use  Access to instrument since Jan 2010  34 different fully-trained operators to date  7 additional people have begun training, but have not yet completed a solo run

33 Ultra-Deep vs. Ultra-Wide Sequencing 2 nd & 3 rd Generation = thousands / millions of sequences per run Cost per run is high ($1000s) Can examine polymorphic target at high depth (ultra-deep) – expensive Can sequence many samples sequenced at the same time (ultra-wide) – cheap

34 Ultra-Deep vs. Ultra-Wide Sequencing Significantly improves sensitivity over traditional Sanger-based sequencing (500x vs 2x coverage)

35 Ultra-Deep vs. Ultra-Wide Sequencing Ultra-deep Ultra-wide Low frequency ARV resistance TCR sequencing Antibody sequencing HLA Typing Allele frequencies SNP detection

36 Multiplexed (Ultra-wide) Amplicon Sequencing Multiplex Identifier MID Tag

37 Methods to increase multiplexing 1.Physically subdividing plate (gasket) 2.Sample specific MID sequence tags 3.Uniquely mixing 5’ & 3’ MID tags PatientMID 1ATCGTAGTCA 2TCCGATCGA 3GTGTAACGT 4CCATGGATC 5TGGATGCAG 6TAGTAGCCA 7GTAGTCTAA 8AACGATGCA 9GCGCTAGCA Patient5' MID3' MID 111 212 313 421 522 623 731 832 933 1. 3. 2.

38 O’Connor lab sequencing projects NHP comprehensive MHC genotyping & allele discovery (amplicons)

39 Importance of MHC Class I  MHC class I molecules dictate immunity to disease  High degree of polymorphism within the MHC class I peptide-binding domain  Specific MHC alleles associated with superior control of HIV infection Source: modified from Yewdell et al., Nature Reviews Immunology 2003 Host Immune Genetics

40 NHP MHC Class I Allele Libraries Total # Alleles in GenBank 663 0 9 156 460

41 NHP MHC Class I Allele Libraries Total # Alleles in GenBank 663 0 9 156 460 Human HLA class I = 5,400 alleles

42 Human HLA vs NHP MHC Class I AC AC B B Human HLA class I

43 Human HLA vs NHP MHC Class I AC AC B B Human HLA class I A1 A1 A2 A2 A4 A4 A3 A3 B1 B1 B2 B2 B3 B3 B4 B4 BN BN A1 A1 A2 A2 A 3 A 4 B1 B1 B2 B2 B3 B3 B4 B4 BN BN Nonhuman primate MHC class I

44 MHC Genotyping Design 568bp amplicon captures highly variable peptide binding region flanked by conserved sequences Amplifies in multiple primate species Longer reads provide better resolution of alleles % MHC Class I Variability 100 80 60 40 20 0 Leader Peptide α 1 Domain α 2 Domain α 3 Domain Cyto- plasmi c Trans- membran e Amino Acid Position FR 568bp Amplicon

45 MHC Genotyping Design 568bp Amplicon Primer = Adapter (A or B) + MID + sequence-specific

46 MHC Genotyping Design 568bp Amplicon Primer = Adapter (A or B) + MID + sequence-specific Within a single nonhuman primate sample:

47 MHC Genotyping Design 568bp Amplicon Primer = Adapter (A or B) + MID + sequence-specific Within an MHC class I amplicon genotyping pool:

48 Roche/454 MHC Workflow Total RNA isolation and cDNA synthesis – RNA isolation ~4 hrs; cDNA synthesis ~2 hrs Primary PCR amplification – plus SPRI purification, quantification, pooling ~3 hrs emPCR – set-up ~1 hr, run ~5.5 hrs Breaking and enrichment – ~3 hrs GS Junior run – set-up ~1.5 hrs; run time ~10 hrs Data processing and analysis – run processing ~2 hrs; – analysis time varies www.454.co m

49 GS Junior Run Metrics – MHC

50 Reads per Sample Sample MIDRead CountSample MIDRead Count Monkey001 1525 Monkey049 49585 Monkey002 2392 Monkey050 50504 Monkey003 31,023 Monkey051 51673 Monkey004 4504 Monkey052 52565 Monkey005 5450 Monkey053 53893 Monkey006 6722 Monkey054 54581 Monkey007 7622 Monkey055 55623 Monkey008 8489 Monkey056 56955 Monkey009 9344 Monkey057 57698 Monkey010 10635 Monkey058 58792 Monkey011 11660 Monkey059 59655 Monkey012 12796 Monkey060 601,203 Monkey013 13653 Monkey061 61428 Monkey014 14731 Monkey062 628 Monkey015 151,342 Monkey063 63391 Monkey016 16628 Monkey064 64663 Monkey017 1776 Monkey065 65411 Monkey018 18481 Monkey066 66386 Monkey019 19503 Monkey067 67625 Monkey020 20633 Monkey068 68637 Monkey021 21573 Monkey069 69367 Monkey022 22463 Monkey070 70391 Monkey023 23390 Monkey071 71585 Monkey024 24723 Monkey072 72808 Monkey025 25739 Monkey073 73594 Monkey026 26560 Monkey074 74391 Monkey027 271,672 Monkey075 75578 Monkey028 28559 Monkey076 76728 Monkey029 29801 Monkey077 77612 Monkey030 30590 Monkey078 78283 Monkey031 31548 Monkey079 79475 Monkey032 32748 Monkey080 80527 Monkey033 33583 Monkey081 8127 Monkey034 34374 Monkey082 82226 Monkey035 35226 Monkey083 83113 Monkey036 36791 Monkey084 84481 Monkey037 37618 Monkey085 8552 Monkey038 38558 Monkey086 86612 Monkey039 39438 Monkey087 87733 Monkey040 40666 Monkey088 88800 Monkey041 41250 Monkey089 89647 Monkey042 42451 Monkey090 901,094 Monkey043 43612 Monkey091 91522 Monkey044 44673 Monkey092 92756 Monkey045 45570 Monkey093 93624 Monkey046 46207 Monkey094 94912 Monkey047 47604 Monkey095 95610 Monkey048 48180 Monkey096 96514

51 Allele Calls & Transcript Profiles % Total Reads MHC Class I Alleles

52 Lymphocyte Specific Expression % Total Reads MHC Class I Alleles

53 ROGER: INSERT ADDITIONAL DATA SLIDES?

54 Same methods applicable to HLA typing We have developed a similar assay to genotype human samples: HLA Class I and DRB loci Cheaper, higher-resolution, and higher- throughput than existing methods Can genotype up to 96 individuals per GS-Jr run

55 High Resolution HLA Genotyping LP α 1 Domain α 2 Domain α 3 Domain CT TM 581-F / 1kb-R bp SBT (Amplicon 2)

56 High-resolution Typing for 40 Reference Cell Lines UW ID#A*B*C* HLA-Ref01A*31:01:02 B*51:01:01 C*15:02:01 HLA-Ref02A*32:01:01 B*38:01:01 C*12:03:01:01/02 HLA-Ref03A*02:16A*03:01:01:01/03B*51:01:01 C*07:04:01C*15:02:01 HLA-Ref04A*24:02:01:01/02LA*26:02B*40:06:01:01/02B*51:01:01C*08:01:01C*14:02:01 HLA-Ref05A*30:01:01 B*13:02:01 C*06:02:01:01/02 HLA-Ref06 A*02:01:01:01/02L/0 3A*02:07B*46:01:01 C*01:02:01 HLA-Ref07A*33:03:01 B*44:03:01 C*14:03 HLA-Ref08A*30:01:01A*68:02:01:01/02/03B*42:01:01 C*1701 HLA-Ref09A*02:06:01A*11:01:01B*15:01:01:01B*35:01:01:01/02C*03:03:01C*04:01:01:01/02/03 HLA-Ref10A*26:01:01 B*08:01:01 C*07:01:01 HLA-Ref11A*02:04 B*51:01:01 C*15:02:01 HLA-Ref12A*03:01:01:01/03 B*47:01:01:01/02 C*06:02:01:01/02 HLA-Ref13A*01:01:01:01 B*57:01:01 C*06:02 HLA-Ref14 A*02:01:01:01/02L/0 3 B*35:03:01 C*12:03:01:01/02 HLA-Ref15 A*02:01:01:01/02L/0 3 B*35:01:01:01/02 C*04:01:01:01/02/03 HLA-Ref16A*34:01:01 B*15:21B*15:35C*04:03C*07:02:01:01/02/03 HLA-Ref17 A*02:01:01:01/02L/0 3 B*15:01:01:01 C*03:04:01:01/02 HLA-Ref18A*01:01:01:01 B*49:01:01 C*07:01:01 HLA-Ref19A*25:01 B*51:01:01 C*01:02 HLA-Ref20A*30:02:01 B*18:01:01:01 C*05:01:01:01/02 HLA-Ref21A*01:01:01:01A*02:05:01B*08:01:01B*50:01:01C*06:02:01:01/02C*07:01:01 HLA-Ref22A*01:01:01:01A*03:01:01:01/03B*07:02:01B*58:01:01C*07:01:01C*07:02:01:01/02/03 HLA-Ref23A*01:01:01A*02:01B*05:801B*07:02C*07:01C*07:02 HLA-Ref24A*01:01:01:01A*24:02:01:01/02LB*39:06:02B*58:01:01C*07:01:01C*07:02:01:01/02/03 HLA-Ref25A*01:01:01:01A*01:37B*35:01:01:01/02B*58:01:01 HLA-Ref26A*03:01:01:01/03 B*07:02:01B*35:01:01:01/02C*04:01:01:01/02/03C*07:02:01:01/02/03 HLA-Ref27A*03:01:01:01/03 B*07:02:01B*35:01:01:01/02C*04:01:01:01/02/03C*07:02:01:01/02/03 HLA-Ref28A*01:01:01:01A*03:01:01:01/03B*35:01:01:01/02B*58:01:01C*04:01:01:01/02/03C*07:18 (701?) HLA-Ref29A*03:01:01:01/03A*24:02:01:01/02LB*35:01:01:01/02B*51:01:04C*04:01:01:01/02/03C*07:04:01 HLA-Ref30 A*02:01:01:01/02L/0 3A*03:01:01:01/03B*07:02:01B*37:01:01C*06:02:01:01/02C*07:02:01:01/02/03 HLA-Ref31A*01:01:01:01A*24:02:01:01/02LB*39:06:02B*58:01:01C*07:01:01C*07:02:01:01/02/03 HLA-Ref32A*24:02:01:01/02L B*07:02:01B*51:01:01C*07:117 HLA-Ref33A*03:01:01:01/03 B*07:02:01B*35:01:01:01/02C*04:01:01:01/02/03C*07:02:01:01/02/03 HLA-Ref34A*03:01:01:01/03A*24:02:01:01/02LB*35:01:01:01/02B*39:06:02C*04:01:01:01/02/03C*07:02:01:01/02/03 HLA-Ref35 A*02:01:01:01/02L/0 3A*24:02:01:01/02LB*07:02:01B*13:02:01C*06:02:01:01/02C*07:02:01:01/02/03 HLA-Ref36A*24:02:01:01/02LA*31:01:02B*07:02:01B*40:01:02C*03:04:01:01/02C*07:02:01:01/02/03 HLA-Ref37 A*02:01:01:01/02L/0 3A*24:02:01:01/02LB*15:01:01:01B*39:06:02C*03:03:01C*07:02:01:01/02/03 HLA-Ref38A*3402A*7401B*801B*1503C*02:10C*701 HLA-Ref39A*2308NA*301B*440301B*5129C*02:02:02C*04 HLA-Ref40 A*02:01:01:01/02L/0 3A*29:02:01B*35:01:01:01/02B*44:03:01C*04:01:01:01/02/03C*16:01:01

57 Example High-Resolution HLA Genotypes with DRB SampleAllele Read s1kbF581F581R1kbRDRB-FDRB-R HIV_114A*36:01122354123 HIV_114A*68:01:011505045505 HIV_114B*41:02:01741624259 HIV_114B*53:01:0122336876139 HIV_114C*04:01:019914521320 HIV_114C*17:01:01 (primer) 4523229 HIV_114DRB1*01:02:01163 8380 HIV_114DRB1*16:02:01127 6562 HIV_114DRB5*02-novel?60. HIV_115A*03:01:01602416713 HIV_115A*11:01:01703216913 HIV_115B*07:02:0112028481232 HIV_115B*51:01:0117753 3536 HIV_115C*07:02:01623015161 HIV_115C*15:02:0110960201910 HIV_115DRB1*04:04:01165 8679 HIV_115DRB1*07:01:01228 114 HIV_115DRB4*01:01:01:0193 7518 HIV_115DRB4*01:03:01:0199 7524 HIV_116A*01:01:011223731495 HIV_116A*02:01:01974017319 HIV_116B*08:01:0121357716322 HIV_116B*15:01:0112921583218 HIV_116C*03:04:0110327432112 HIV_116C*07:01:011144622415 HIV_116DRB1*03:01:01471 244227 HIV_116DRB1*04:01:01429 221208 HIV_116DRB3*01:01:02137 7463 HIV_116DRB4*01:03:01:01176 10175 SampleAlleleReads1kbF581F581R1kbRDRB-FDRB-R HIV_117A*26:01:0116724744029 HIV_117A*29:02:019624312417 HIV_117B*44:03:01 (putative)286112535962 HIV_117B*44:10 (putative)2101135146. HIV_117C*04:01:01245381302651 HIV_117 DRB1*03:01:01173 9479 HIV_117DRB1*07:01:01171 8190 HIV_117DRB3*02:02:0150 25 HIV_117DRB4*01:03:01:0144 2915 HIV_118A*02:01:0111733462414 HIV_118A*23:01:0115642613914 HIV_118B*40:01:0211313503515 HIV_118B*44:03:0120651816311 HIV_118C*03:04:018474715 HIV_118C*14:0314228613122 HIV_118DRB1*04:01:01151 8071 HIV_118DRB1*10:01:01195 9699 HIV_118DRB4*01:03:01:0157 3324 HIV_119A*29:01:01:0136137106 HIV_119A*68:01:02733612205 HIV_119B*07:05:01481211718 HIV_119B*44:02:01:01864115264 HIV_119C*05:01:0147255107 HIV_119C*15:05:01/0263261511 HIV_119DRB1*04:04:01233 89144 HIV_119DRB1*07:01:01250 105145 HIV_119DRB4*01:03:01:0177 3344

Download ppt "Welcome to UW-Madison, the WNPRC, and O’Connor Lab! MHC Genotyping Workshop November 7 th – 11 th, 2011 Madison, Wisconsin."

Similar presentations

Next-Generation Sequencing: Methodology and Application

Next-Generation Sequencing: Methodology and Application
Virus discovery-454 sequencing

Virus discovery-454 sequencing
Considerations for Analyzing Targeted NGS Data HLA

Considerations for Analyzing Targeted NGS Data HLA
 Sequencing technology › Roche/454 GS-FLX (‘454’) › Illumina  Prokaryotic profiling › De novo genome sequencing › Metagenomics › SNP profiling › Species.

 Sequencing technology › Roche/454 GS-FLX (‘454’) › Illumina  Prokaryotic profiling › De novo genome sequencing › Metagenomics › SNP profiling › Species.
© 2010 LabKey Software Managing Next Generation Sequencing and Multiplexed Genotyping Data Using Open Source LabKey Server Adam Rauch

© 2010 LabKey Software Managing Next Generation Sequencing and Multiplexed Genotyping Data Using Open Source LabKey Server Adam Rauch
Next–generation DNA sequencing technologies – theory & practice

Next–generation DNA sequencing technologies – theory & practice
ANIMAL MODELS HIV Cure Research Training Curriculum The HIV CURE training curriculum is a collaborative project aimed at making HIV cure research science.

ANIMAL MODELS HIV Cure Research Training Curriculum The HIV CURE training curriculum is a collaborative project aimed at making HIV cure research science.
Bioinformatics Lectures at Rice

Bioinformatics Lectures at Rice
Introduction  Human leukocyte antigen (HLA) is the major histocompatibility complex (MHC) in humans  Group of genes ('superregion') on chromosome 6.

Introduction  Human leukocyte antigen (HLA) is the major histocompatibility complex (MHC) in humans  Group of genes ('superregion') on chromosome 6.
Next-generation sequencing

Next-generation sequencing
The 454 and Ion PGM at the Genomics Core Facility Dr. Deborah Grove, Director for Genetic Analysis Genomics Core Facility Huck Institutes of the Life Sciences.

The 454 and Ion PGM at the Genomics Core Facility Dr. Deborah Grove, Director for Genetic Analysis Genomics Core Facility Huck Institutes of the Life Sciences.
Canadian Bioinformatics Workshops

Canadian Bioinformatics Workshops
Greg Phillips Veterinary Microbiology gregory@iastate.edu.

Greg Phillips Veterinary Microbiology
HIV/AIDS as a Microcosm for the Study of Evolution.

HIV/AIDS as a Microcosm for the Study of Evolution.
Laboratory of Experimental Virology Virus Discovery 454 sequencing Michel de Vries

Laboratory of Experimental Virology Virus Discovery 454 sequencing Michel de Vries
1 Characterization, Amplification, Expression Screening of libraries Amplification of DNA (PCR) Analysis of DNA (Sequencing) Chemical Synthesis of DNA.

1 Characterization, Amplification, Expression Screening of libraries Amplification of DNA (PCR) Analysis of DNA (Sequencing) Chemical Synthesis of DNA.
What Can You Do With qPCR?

What Can You Do With qPCR?
An Introduction to the HIV Problem Space Oakwood University: Faculty Quantitative Institute Aug. 10–12, 2009.

An Introduction to the HIV Problem Space Oakwood University: Faculty Quantitative Institute Aug. 10–12, 2009.
Next Generation DNA Sequencing Platforms: Evolving Tools for

Next Generation DNA Sequencing Platforms: Evolving Tools for
Diabetes and Endocrinology Research Center The BCM Microarray Core Facility: Closing the Next Generation Gap Alina Raza 1, Mylinh Hoang 1, Gayan De Silva.

Diabetes and Endocrinology Research Center The BCM Microarray Core Facility: Closing the Next Generation Gap Alina Raza 1, Mylinh Hoang 1, Gayan De Silva.

Similar presentations

Ads by Google