Holotype HLA
Agenda §
Introducing... Holotype HLA
§
Holotype HLA Assay Advantages
§
HLA Twin So8ware Overview (live demo)
§
Locus Coverage & Throughput Scalability (within demo)
§
Summary & Benefits
§
Q&A
Introduc6on to Holotype HLA™ Holotype HLA is a combinaFon Assay and So8ware product for the comprehensive gene amplificaFon of mulFple HLA loci, and sequencing on the Illumina MiSeq.
NGS Assay + NGS So8ware
Omixon Holotype HLA – 5 loci (Assay) 5 loci – HLA-‐A, B, C, DQB1 and DRB1
§ § § § §
X4 – 16/5 96 Indexes 5+1 configuraFon (per locus indexing)
§
2 technical replicates per amplicon 2 genotyping algorithms
§
§ §
§
X2 – 96/5 96 Indexes Fully pooled configuraFon (per sample indexing) 1 technical replicate per amplicon 2 genotyping algorithms
Omixon Holotype HLA – 7 loci (Assay) 7 loci – HLA-‐A, B, C, DRB1, DPB1, DQA1 and DQB1
§ § § § §
X2 – 24/7 24 Indexes Fully pooled configuraFon (per sample indexing)
§
1 technical replicate per amplicon 2 genotyping algorithms
§
§ §
§
X2 – 96/7 96 Indexes Fully pooled configuraFon (per sample indexing) 1 technical replicate per amplicon 2 genotyping algorithms
LR-‐PCR Amplifica6on Strategy Full gene characteriza6on HLA-‐A, B, C, and DQA1 (Example is HLA-‐B) 5’ UTR 1
2
3
4
5
6
7
3’ UTR
HLA-‐DQB1 5’ UTR 1
2
3
4
5
6 3’ UTR
Key region characteriza6on HLA-‐DRB1 5’ UTR 1
2
3
4
5
6 3’ UTR
HLA-‐DPB1 5’ UTR 1
2
3
4
5 3’ UTR
NGS Library Prepara6on Simplicity gDNA FragmentaFon
End Repair
Adaptor LigaFon
Paired-‐End Sequencing
Single-‐End Reads Read 1 Template
Paired-‐End Reads Read 1 Template
Read 2
Sequence from the paired reads originate from the same template, and therefore are phased with each other
Effect of Varying Insert Sizes among Paired Reads Read Length 250 bases Read Length 250 bases
Overlap of 100 bases
Fragment Length 400 bp Read Length 250 bases
Read Length 250 bases
No Overlap
Fragment Length 500 bp Read Length 250 bases
Read Length 250 bases
No sequence for 100 bases
Fragment Length 600 bp
Op6mized Size Selec6on Fragment Size
Number of read pairs referring to fragments within the specified length interval
Long Distance Phasing B*07:02:01
T
T
A G
T A
C
T
B*41:01
C
C
T C
C G
T
G
Allelic Balance
The opFmized assay design achieves allelic balance across all loci for accurate HLA allele determinaFon without bias towards easy-‐to-‐sequence genomic regions.
Even Coverage Across a locus
Across loci within a sample
Between samples
Omixon HLA Twin™ (So]ware) §
Preconfigured Holotype HLA-‐specific sedngs
§
Automated genotyping a8er MiSeq run
§
Two algorithms for determining HLA genotypes – Consensus Genotyping (Assembly) – StaFsFcal Genotyping (Alignment to IMGT/HLA)
§
Traffic light system for data interpretaFon and workflow management PASSED WARNING FAILED
The Traffic Light System (Overview)
The Traffic Light System
Live Demo §
Live Demo of Holotype HLA Twin™
Benefits of Holotype HLA Assay §
LR-‐PCR for fully characterized loci
§
Easy Library PreparaFon
§
Variable-‐insert paired-‐end sequencing for fully phased allele variaFon
§
High-‐throughput NGS on a MiSeq
§
Unambiguous genotyping
§
No reflexive tesFng
Benefits of HLA Twin So]ware §
Accurate genotyping with two orthogonal algorithms
§
No manual intervenFon for good quality data
§
InformaFve quality control metrics for confidence in genotyping results
§
Traffic Light System for easy interpretaFon – Novel allele detecFon – Null alleles always resolved (splice variant and intronic)
§
Export genotypes or consensus sequence for reporFng
Holotype HLA vs Compe6tors Competing Products
Holotype HLA
Occasional allele dropout
Very rare allele dropout
6+ hours hands on time
About 4 hours hands on time
Up to 24 samples per run
96 samples per run
Uneven coverage, dips in coverage at key exons
Deep & even coverage across every exon for each locus
Allele imbalance
Balanced coverage depth for both alleles at each locus
One algorithm, requires technician for every allele call
Fully automated genotyping with two algorithms
High rates of ambiguity
0.2% ambiguity
The Omixon Website
The Omixon Academy Omixon'('Academy'
Ques6ons?
[email protected] +1 (617) 500 0790
Introducing... Holotype HLA
§
Holotype HLA Assay Advantages
§
HLA Twin So8ware Overview (live demo)
§
Locus Coverage & Throughput Scalability (within demo)
§
Summary & Benefits
§
Q&A
Introduc6on to Holotype HLA™ Holotype HLA is a combinaFon Assay and So8ware product for the comprehensive gene amplificaFon of mulFple HLA loci, and sequencing on the Illumina MiSeq.
NGS Assay + NGS So8ware
Omixon Holotype HLA – 5 loci (Assay) 5 loci – HLA-‐A, B, C, DQB1 and DRB1
§ § § § §
X4 – 16/5 96 Indexes 5+1 configuraFon (per locus indexing)
§
2 technical replicates per amplicon 2 genotyping algorithms
§
§ §
§
X2 – 96/5 96 Indexes Fully pooled configuraFon (per sample indexing) 1 technical replicate per amplicon 2 genotyping algorithms
Omixon Holotype HLA – 7 loci (Assay) 7 loci – HLA-‐A, B, C, DRB1, DPB1, DQA1 and DQB1
§ § § § §
X2 – 24/7 24 Indexes Fully pooled configuraFon (per sample indexing)
§
1 technical replicate per amplicon 2 genotyping algorithms
§
§ §
§
X2 – 96/7 96 Indexes Fully pooled configuraFon (per sample indexing) 1 technical replicate per amplicon 2 genotyping algorithms
LR-‐PCR Amplifica6on Strategy Full gene characteriza6on HLA-‐A, B, C, and DQA1 (Example is HLA-‐B) 5’ UTR 1
2
3
4
5
6
7
3’ UTR
HLA-‐DQB1 5’ UTR 1
2
3
4
5
6 3’ UTR
Key region characteriza6on HLA-‐DRB1 5’ UTR 1
2
3
4
5
6 3’ UTR
HLA-‐DPB1 5’ UTR 1
2
3
4
5 3’ UTR
NGS Library Prepara6on Simplicity gDNA FragmentaFon
End Repair
Adaptor LigaFon
Paired-‐End Sequencing
Single-‐End Reads Read 1 Template
Paired-‐End Reads Read 1 Template
Read 2
Sequence from the paired reads originate from the same template, and therefore are phased with each other
Effect of Varying Insert Sizes among Paired Reads Read Length 250 bases Read Length 250 bases
Overlap of 100 bases
Fragment Length 400 bp Read Length 250 bases
Read Length 250 bases
No Overlap
Fragment Length 500 bp Read Length 250 bases
Read Length 250 bases
No sequence for 100 bases
Fragment Length 600 bp
Op6mized Size Selec6on Fragment Size
Number of read pairs referring to fragments within the specified length interval
Long Distance Phasing B*07:02:01
T
T
A G
T A
C
T
B*41:01
C
C
T C
C G
T
G
Allelic Balance
The opFmized assay design achieves allelic balance across all loci for accurate HLA allele determinaFon without bias towards easy-‐to-‐sequence genomic regions.
Even Coverage Across a locus
Across loci within a sample
Between samples
Omixon HLA Twin™ (So]ware) §
Preconfigured Holotype HLA-‐specific sedngs
§
Automated genotyping a8er MiSeq run
§
Two algorithms for determining HLA genotypes – Consensus Genotyping (Assembly) – StaFsFcal Genotyping (Alignment to IMGT/HLA)
§
Traffic light system for data interpretaFon and workflow management PASSED WARNING FAILED
The Traffic Light System (Overview)
The Traffic Light System
Live Demo §
Live Demo of Holotype HLA Twin™
Benefits of Holotype HLA Assay §
LR-‐PCR for fully characterized loci
§
Easy Library PreparaFon
§
Variable-‐insert paired-‐end sequencing for fully phased allele variaFon
§
High-‐throughput NGS on a MiSeq
§
Unambiguous genotyping
§
No reflexive tesFng
Benefits of HLA Twin So]ware §
Accurate genotyping with two orthogonal algorithms
§
No manual intervenFon for good quality data
§
InformaFve quality control metrics for confidence in genotyping results
§
Traffic Light System for easy interpretaFon – Novel allele detecFon – Null alleles always resolved (splice variant and intronic)
§
Export genotypes or consensus sequence for reporFng
Holotype HLA vs Compe6tors Competing Products
Holotype HLA
Occasional allele dropout
Very rare allele dropout
6+ hours hands on time
About 4 hours hands on time
Up to 24 samples per run
96 samples per run
Uneven coverage, dips in coverage at key exons
Deep & even coverage across every exon for each locus
Allele imbalance
Balanced coverage depth for both alleles at each locus
One algorithm, requires technician for every allele call
Fully automated genotyping with two algorithms
High rates of ambiguity
0.2% ambiguity
The Omixon Website
The Omixon Academy Omixon'('Academy'
Ques6ons?
[email protected] +1 (617) 500 0790
