1. 16-point HLA Typing with Long Amplicon Analysis v2
Brett Bowman
September 14th, 2016

2. What is 16-point HLA Typing?
“Classical” MHC Proteins
3 CD8-presenting complexes
“MHC Class I”
HLA-A, B, C
1 Gene per AS
3 CD4-presenting complexes
“MHC Class II”
HLA-DP, DQ, DR
2 Genes Per AS 2x 1x 1x

3. AGENDA Long Amplicon Analysis (LAAv2) 16-point HLA Sequencing Results
Overview
Improvements in LAAv2 for Complex HLA Typing
16-point HLA Sequencing Results
Experimental Design
Typing Results
Sequence Quality

4. Long Amplicon Analysis WorkFlow – overview
Subread Filtering
Coarse Clustering
Waterfall Clustering
Fine Phasing
Consensus
Post-Processing Filters
Outputs
Subread Filtering
Coarse Clustering
Waterfall Clustering
Fine Phasing
Consensus
Post-Processing Filters
Outputs
Subread Filtering
Coarse Clustering
Waterfall Clustering
Fine Phasing
Consensus
Post-Processing Filters
Outputs
Sample
Locus
Allele
Clean-Up
Report

5. Long Amplicon Analysis WorkFlow – Coarse & WATERFALL Clustering
Subread Filtering
Coarse Clustering
Waterfall Clustering
Fine Phasing
Consensus
Post-Processing Filters
Outputs
Filtered Subreads
Clustering Subreads
Waterfall Subreads
Cluster #1
Cluster #2
Cluster #3

6. Long Amplicon Analysis WorkFlow – Fine Phasing & Consensus
Subread Filtering
Coarse Clustering
Waterfall Clustering
Fine Phasing
Consensus
Post-Processing Filters
Outputs

7. Results
Sample Experimental Designs & Results

8. What is 16-point HLA Typing?
HLA Complete Panel
Exon sequence only
2-step PCR
9 outer PCR
~45 inner PCR
Phasing by inference
3-day turn around
~QV30
Experimental Design
Re-use outer primers
3.1~5.8 kb
Active-site genomic
7-loci fully phased
>QV50

9. Long Amplicon AnALYSIS – FULL PANEL RESULTS
Sequence
gLen
gType
gPctId
nMis
Indel
cLen
cType
cPctId
Type
Cluster0_Phase0_NumReads192
3152
A*34:01:01
100
1093
Cluster1_Phase0_NumReads194
3406
B*15:02:01
99.94 2
1045
B*15:21
Cluster2_Phase1_NumReads203
3395
B*40:01:02
Cluster2_Phase0_NumReads184
3460
C*04:03:01
1096
Cluster3_Phase1_NumReads228
3445
C*12:03:01:01
C*12:03:01
Cluster3_Phase0_NumReads215
4839
DPA1*01:03:01:04
96.88 52
601
DPA1*02:02:02
Cluster4_Phase0_NumReads234
5801
DPB1*03:01:01
99.31 39 1
657
DPB1*01:01:01
Cluster5_Phase1_NumReads178
4572
DPB1*04:02:01:02
99.47 18 6
Cluster6_Phase1_NumReads201
5743
DQA1*01:02:01:01
99.97
613
DQA1*01:02:01
Cluster5_Phase0_NumReads240
5692
DQA1*03:03:01
99.95 3
Cluster7_Phase1_NumReads236
3768
DQB1*03:05:01
99.28 19 8
618
DQB1*04:02:01
Cluster6_Phase0_NumReads216
3855
DQB1*05:01:01:01
99.52 16
DQB1*05:02:01
Cluster8_Phase1_NumReads187
5105
DRB1*04:03:01
99.05 17 31
584
DRB1*04:05:01
Cluster7_Phase0_NumReads217
3893
DRB1*15:01:01:03
DRB1*15:02:01
Sequence
gLen
gType
gPctId
nMis
Indel
cLen
cType
cPctId
Type
Cluster0_Phase0_NumReads195
3138
A*11:01:01:01
100
1093
A*11:01:01
Cluster1_Phase1_NumReads173
3153
A*74:02:01:02
99.84 5
A*74:01
Cluster1_Phase0_NumReads189
5778
B*07:02:01
1045
Cluster2_Phase1_NumReads179
3398
B*53:01:01
Cluster2_Phase0_NumReads212
3424
C*04:01:01:01
1096
C*04:01:01
Cluster3_Phase1_NumReads222
3429
C*07:02:01:03
C*07:02:01
Cluster4_Phase1_NumReads188
4830
DPA1*01:03:01:02
97.06
103 40
601
DPA1*02:01:01
Cluster3_Phase0_NumReads230
4857
DPA1*01:03:01:04
DPA1*01:03:01
Cluster5_Phase3_NumReads200
4574
DPB1*02:01:02
99.58 18 1
107
DPB1*01:01:01
Cluster4_Phase0_NumReads186
5800
DPB1*03:01:01
99.48 28 2
657
DPB1*107:01
Cluster5_Phase2_NumReads225
4569
DPB1*04:01:01:01
99.98
Cluster5_Phase1_NumReads220
5872
99.86 8
DPB1*04:01:01
Cluster5_Phase0_NumReads201
5751
DQA1*01:02:01:04
99.93 3
613
DQA1*01:02:01
Cluster6_Phase1_NumReads228
5691
DQA1*03:03:01
Sequence
gLen
gType
gPctId
nMis
Indel
cLen
cType
cPctId
Type
Cluster0_Phase0_NumReads176
3138
A*01:01:01:01
100
1093
A*01:01:01
Cluster1_Phase1_NumReads177
3152
A*02:01:01:01
A*02:01:01
Cluster1_Phase0_NumReads228
3395
B*08:01:01
1045
Cluster2_Phase1_NumReads189
B*40:01:02
Cluster2_Phase0_NumReads171
3422
C*03:04:01:01
1096
C*03:04:01
Cluster3_Phase1_NumReads200
3430
C*07:01:01:01
C*07:01:01
Cluster3_Phase0_NumReads175
4858
DPA1*01:03:01:02
602
DPA1*01:03:01
Cluster4_Phase0_NumReads193
4568
DPB1*04:01:01:01
DPB1*01:01:01
Cluster5_Phase1_NumReads210
5875
99.95 1 2
657
DPB1*04:01:01
Cluster6_Phase1_NumReads210
5691
DQA1*03:01:01
613
Cluster5_Phase0_NumReads235
5799
DQA1*05:01:01:02
610
DQA1*05:01:01
Cluster6_Phase0_NumReads239
4114
DQB1*02:01:01
99.98
618
Cluster7_Phase1_NumReads210
3775
DQB1*03:02:01
99.92
Cluster7_Phase0_NumReads175
3686
DRB1*03:01:01:01
584
DRB1*03:01:01

10. Long Amplicon AnALYSIS – Class I Results
HLA Class I
3 genes
3.1~3.4 kb gDNA
~1.5 kb active-site
3 Sanger rxns ea
~QV30
Experimental Design
45x samples
All Class I genes
Complete genomic
Fully phased
>QV50

11. Introducing – Loci Analysis
LociAnalysis -vv -n 16 -o OUTPUT_DIR --combineLoci DPB:DPB1:DPB ignoreLoci DPB1,DPB2,H --minLengthByLocus DRB4: maxLengthByLocus DRB4: maxClusteringReadsByLocus DRB1: maxReadsByLocus DRB1: DATABASE_FOLDER SUBREADS.BAM