DNA based HLA typing methods By: Yadollah Shakiba, MD, PhD
MHC Region MHC expression: Class I: All nucleated cells and platelets Class II: Antigen presenting cells
Nomenclature of HLA Alleles
Assigned as of April 2016 HLA class I Alleles Gene A B C Allelesl 3,399399 4,242 2,950 Proteins 2,396 3,131 2,089 Nulls 157 134 109
DNA Based HLA typing SSP SSOP Sanger SBT Next generation sequencing
PCR SSP maximum, GA, CT, TT; strong, CC; medium, AA, GG; Weak, CA, GT; AT, GC
HLA-A Exon 3 Sequences
HLA-A*01 PCR-SSP Internal control: GH F-TGCCAAGTGGAGCACCCAA R-GCATCTTGCTCTGTGCAGAT 796 bp HLA-A*01 Allele group specific primer Forward: TCTCACACCATCCAGATA Reverse: ACCGGCCCTCCAGGTAGA 220 bp
HLA-A*36 PCR-SSP Mix A*36 F1: GTGGGAGGCGGTCCATGC 36:01 36:05 R1:AGGTATCTGCGGAGCCAC 03:187/11:155/31:62 PCR product 80 bp
HLA-A*36 A PCR-SSP primer blast
Applications of PCR-SSP Rapid HLA screening (Low resolution) Allele group amplification before SBT HLA and disease study (AS: B*27, RA:DRB1*04, narcolepsy: DQB1*06:02, 02 Bh Behçetdisease: B*51) High resolution typing (sub-typing)
The more alleles The more problems!!
Sequence Based Typing (SBT) Sanger SBT SBT by NGS
Sanger Sequencing The chain-termination method invented by Fred Sanger. DNA polymerase always adds new bases to the 3 end of a primer that is base-paired to the template DNA. dideoxy nucleotides (ddntps)
Manual Sanger DNA sequencing
Sanger platforms
Recent advances in Sanger sequencing (automated) ddntp's with fluorescent markers we can mix all four types together and do one reaction instead of four Theresultingfragmentsarestillof are still different lengths Separate fragments via electrophoresis in gelfilled capillary tubes. Automatic sequencing machines read output
Sanger HLA Sequencing
HLA typing by automated SBT Unrelated HSCT Cord blood transplantation (DRB1) HSCT from sibling with ambiguities Kidney transplant? HLA match maker
NGS platforms Illumina MiSeq Ion Torrent PGM Pacific Biosciences RSII Illumina MiniSeq ThermoFisher 26 Ion S5 PacBio Sequel
Illumina sequencing Amplification lf strategies Amplicon sequencing Target Library Amplify Shotgun sequencing Sequence Sequence Analyse
HLA amplicon quantification Target Pooling UT R 1 2 3 4 5 6 7 8 UT R Gelelectrophoresis Qubit Fragment Equimolar amplicon pooling Adapter e.g. 96 samples, 11 loci => 96 amplicon pools Index Quant. Amplify NGSgo AmpX amplicon Concentration (ng/µl) #1 #2 #3 Average concentration (ng/µl) Amplicon length (bp) Amplicon volume (µl) in pool HLA A 351 383 369 368 3100 4,0 HLA B 295 331 311 319 3400 5,1 HLA C 265 306 270 280 3400 5,8 HLA DRB1 355 452 408 405 4800 5,6 HLA DQB1 435 458 444 446 4100 3 Sequence Analyse
Pooling of HLA amplicons Target Pooling Fragment Adapter Index Quant. Amplify Sequence Analyse Illumina MiSeq Workflow
HLA library preparation Reaction #1: Enzymatic fragmentation, end repair, A tailing
library preparation Step 2: Ligation of Illumina compatible adapter Illumina MiSeq Workflow
Paired end Sequencing Illumina approach 300 nt P5 ID Rd1 SP DNA fragment Rd2 SP ID P7 G T C C R A G A G G G - - - C C G C G G G M G C C.... A................ A....... G................ C...
Paired end Sequencing Illumina approach In silico linking of information for larger fragment sizes 150 nt 150 nt P5 ID Rd1 SP DNA fragment Rd2 SP ID P7 G T C C R A G A G G G - - - C C G C G G G M G C C.... A................ A....... G................ C...
DNA clean up and size selection Target Pooling Fragment Adapter Index Quant. Crowding agents (20% PEG, 2.5M NaCl) Beads : DNA ratio > affects size selection Amplify Sequence Analyse Illumina MiSeq Workflow
Indexing PCR Target Pooling Fragment Adapter Index Quant. Amplify Sequence Analyse Illumina MiSeq Workflow
HLA library preparation Dual indexing PCR: insert index sequencing primer binding sites flow cell attachment sites index Illumina MiSeq Workflow
Indexing PCR: Dual Indexing Target Pooling Fragment Adapter Index Quant. Amplify Sequence Analyse Illumina MiSeq Workflow
Dual indexing 4x24 rxn 2x96 rxn 1x384 rxn 70 70 70 70 70 70 70 70 70 71 71 71 1 2 3 4 5 6 7 8 9 0 1 2 50 1 50 2 50 3 N 50 IndX kits: 4 1. 50 One adapter 5 50 6 50 7 50 8 50 9 51 0 51 1 51 2 51 2. Panel of indices (Illumina specific) Combination of indices sufficient for dual indexing of4x24 4x24, 2x96, or1x384libraries 71 3 71 4 71 5 71 6 71 7 71 8 71 9 72 0 72 1 72 2 72 3 72 4 Illumina MiSeq Workflow
HLA library pooling Target Pooling LIB 1 LIB 2 LIB 3 LIB 384 Fragment Adapter 1500 bp 1000 bp 500 bp Library > 400 bp Index Quant. Amplify 100 bp Sequence Analyse Illumina MiSeq Workflow
Agrose gel electrophoresis Good quality DNA fragments with a size range of 400 800 bp
Library quantification with KAPA P5 P7 Target Pooling Fragment Adapter Index Quant. 95 o C 95 o C Amplify Sequence 60 o C Analyse Illumina MiSeq Workflow
Library quantification with KAPA Target Pooling Fragment Adapter Index Quant. Amplify Sequence Analyse Illumina MiSeq Workflow
MiSeq run Target Denature library Prepare Sample Sheet Load denatured library on MiSeq cartridge Pooling Fragment Adapter Index Quant. Amplify Sequence 300 cycle kit run => 16 24 hours Analyse
MiSeq reagent kit Box 1 ( 20 C): 1) Pre filled ready to use reagent cartridge 2) Tb Tube of HT 1 buffer 1 2 3 4 Box 2 (+4 C): 3) Container with flow cell 4) Incorporation buffer
Selection of MiSeq reagent cartridge MiSeq Reagent kit Kit size (cycles) Differences between cartridges are: MiSeq Reagent 50, 300, Kit v2 500 MiSeq Reagent 150, 600 quality Kit v3 Reagents Read length and quality Run time Costs Recommended for NGSgo => 300 cycle, v2
Paired end sequencing strategies 300 vs. 500 cycle kit 2x150 bp P5 ID Rd1 SP DNA fragment (~400 bp) Rd2 SP ID P7 NGSengine: optimal pairs 2x250 bp P5 ID Rd1 SP DNA fragment (~400 bp) Rd2 SP ID P7
Three MiSeq flow cell types Standard Micro Nano Standard Micro Nano SURFACE: Top Bottom SURFACE: Top Bottom SURFACE: Top Bottom 28 tiles 8 tiles 2 tiles Same DNA input concentration and same chemistry Different number of tiles for imaging g=> different read output
Flow cell guidelines
Summary MiSeq chemistry Target generation Library preparation p Clonal amplification Sequencing Data analysis Illumina MiSeq Chemistry
Summary MiSeq chemistry Target generation Library preparation p Clonal amplification Sequencing Data analysis Illumina MiSeq Chemistry
Sequencing data A image C image G image T image Illumina MiSeq Chemistry
Ion torrent Workflow Target generation Library preparation NGSgo reagents Clonal amplification Enrichment ISPs Ion One Touch 2 / Ion Chef Sequencing Ion Torrent tpgm Data analysis NGSengine Ion Torrent workflow
Target generation Target generation UT R 1 2 3 4 5 6 7 8 UT R HLA A (3.1 kb) Library preparation UT UT HLA B (3.4 kb) R UT R 1 2 3 4 5 6 7 1 2 3 4 5 6 7 8 R UT R HLA C (3.4 kb) UT R 1 2 3 4 5 6 UT R HLA DRB1 (3.7 4.8 kb) Amplified exon Exon not amplified UT R UT R UT R 1 2 3 4 5 6 1 2 3 4 5 1 2 3 4 UT R UT R UT R HLA DQB1 (3.7 4.1 kb) HLA DPB1 (5.0 & 5.7 kb) HLA DPA1 (4.7 kb) UT R 1 2 3 4 UT R HLA DQA1 (5.4 5.8 kb) UT R 1 2 3 4 5 6 UT R HLA DRB3 (3.8 kb) UT R 1 2 3 4 5 6 UT R HLA DRB4 (0.4 & 1.3 kb) UT R 1 2 3 4 5 6 UT R HLA DRB5 (4.0 kb) Ion Torrent workflow
Pooling of HLA amplicons Target generation Library preparation Measurement with ihqubit Equimolar pooling of HLA amplicons for each sample Ion Torrent workflow
Library preparation Fragmentation, end repair and da tailing Adapter ligation X adapter contains barcode Ion Torrent workflow
Library preparation LIB 1 LIB 2 LIB 3 LIB 24 Equivolume pooling Size selection with SPRI beads Ion Torrent workflow
Library preparation 10 kb 3 kb 1kb 500 bp Size of generated fragments > 400 bp Quantification by KAPA, qpcr Using adapter specific primers 9 pm of final library pool needed Ion Torrent workflow
Workflow Target generation Library preparation Clonal amplification Enrichment ISPs Ion One Touch 2 Ion Chef Sequencing Data analysis Ion Torrent workflow
Clonal amplification DNA fragments bind ion sphere particles (ISPs) with P adapter. Amplification of bound DNA fragments. Biotinylation of X adapter Quality control: %ISPs covered with DNA QC kit for Qubit Ion Torrent workflow
Template preparation systems Ion One Touch 2 System Ion Chef System Clonal amplification system Performs all steps from clonal Enrichment system amplification to chip loading Chip loading manually Can perform two chips simultaneously Ion Torrent workflow
Clonal amplification Ion Torrent workflow
Clonal amplification Ion Torrent workflow
Enrichment ISPs Streptavidin coated magnetic beads bind to biotinylated DNA Polyclonal ISPs are not removed Empty ISPs are removed Ion Torrent workflow
Enrichment ISPs Touch pad Mechanical arm Pipet tip loader Strip (flanked by magnets) Ion Torrent workflow
Workflow Target generation Library preparation Clonal amplification Enrichment ISPs Sequencing Ion Torrent tpgm Data analysis Ion Torrent workflow
Ion Torrent sequencers Ion Proton Ion PGM Ion S5 Ion Torrent workflow
Ion Torrent sequencers Nitrogen Hands on Capacity Mean supply work read length Ion Yes 1 hour High 200 bp Yes Proton Ion PGM Yes 1 hour Low 300 bp Yes Ion S5 No 5 minutes Very high 300 bp NGSgo compati ble Yes Ion Torrent workflow
Chip loading Ion 314 Ion 316 Ion 318 30 Mb 100 Mb 400 550 thousand reads per run 300 Mb 1 Gb 2 3 million reads per run 600 Mb 2 Gb 4 5,5 million reads per run 6samples 5 loci 24 samples 5 loci 24 samples 11 loci Ion Torrent workflow
Chip loading Incubation with sequence primer sequence polymerase Each ISP fits in a hole on chip Amount of loaded ISPs measured by loading density (%) Ion Torrent workflow
Sequencing A flow is the event of exposing the chip to one of the 4 dntps, followed by a wash step The flows of all 4 dntps is called a cycle T A C G is one cycle Ion Torrent workflow
Data analysis: Torrent Browser Ion Torrent Workflow
Data analysis with NGSengine Target Pooling Fragment Adapter Index Quant. Amplify Sequence Analyse
NGSengine analysis Align reads with IMGT/HLA Highly polymorph Phase data Determine cis trans Perform typing Determine the matching genotype(s) New allele Illumina workflow
Sample overview: Analyze NGSengine
Sample overview: Determine locus NGSengine
Sample overview: Aligning reads NGSengine
Sample overview: Phasing NGSenginer
Sample overview: Typing NGSenginer
Sample overview: Analysis finished NGSengine
Analyzed data: Overview NGSengine
Analyzed data: Gene NGSengine
Analyzed data: Coverage Coverage Read Depth NGSengine
Analyzed data: Phasing NGSengine
Analyzed data: Matching alleles NGSengine
Analyzed data: Read overview NGSengine
Quality Statistics: Percentage most frequent base call versus rest NGSengine
Quality Statistics: Percentage most frequent base call versus rest Homozygous 2 alleles ~50% Noise 87
Analyzed data: Genotype Ranking NGSengine
Analyzed ddt data:reporting ti NGSengine