1. Implementation Insights
Eric T. Weimer, PhD, D(ABMLI)

2. CONFLICT OF INTEREST I have financial relationship(s) with:
Advisory Board, Illumina

3. NGS Implementation at UNC
Received ASHI accreditation in August 2015
Molecular Genetics lab has 2 MiSeqs and NextSeq
Evaluated in-house, Illumina, GenDx, and Omixon reagents
Clinically HLA typed ~1400 samples
Validated assay change in May 2016
Validated several software iterations
Developed automation script for Tecan Freedom EVO NGS platform

4. Why TruSight HLA? Low cost to implement
Availability of instrumentation
Other vendors require additional instrumentation
One vendor for ALL NGS reagents
Availability of HLA loci being reported
At the time of evaluation, only TruSight HLA & GenDx had necessary loci
Inclusion of software with purchase of reagents
Overall, ease of translation with current workflow and familiarity with software
UNC previously used Conexio Assign SBT software
Responsiveness of Illumina support

5. NGS Workflow at UNC Manually prepare NGS libraries
No major issues with contamination
Majority of HLA typing at UNC is performed by NGS
HSCT patients, related and unrelated donors
Initial and confirmatory
Report: HLA-A, -B, -C, -DRB1, -DQB1, -DPB1
Solid-organ patients and donors
Store high-resolution HLA typing
Report: Serologic equivalent; HLA-A, -B, -C, - DRB1/3/4/5, -DQB1, -DPB1, Bw4/Bw6
Disease association (Celiac disease)
Confirmatory decease donor typing

6. UNC NGS Process UNC has always prepared NGS libraries manually
Known HLA typed samples used throughout the entire process and rotated for index QC (library preparation control)
Spike-in of PhiX (sequencing control)
Since switching to the v2 assay:
2 technologist used for pipetting
1 technologist for 1.5 hours hands-on
1 technologist for 3 hours hands-on
Typically start preparation by 9am and load MiSeq by 2pm
Sequencing completed within 19 hours (Micro flow cell)
Data analysis
UNC policy: 2 technologist reviews
1st reviewer, reviews software for flags, rare alleles, internal control, and HLA associations
2nd reviewer, confirms1st review
Integrated with HistoTrac (SystemLink) for data import and report generation

7. NGS Improvement of Turn-around-time (TAT)
Initial transition to NGS using v1 assay
Reduced TAT by 4 days compared to Sanger sequencing
Reduced ambiguity resolution
Transition to v2 assay
Reduced TAT by ~9 days
Further reduction in ambiguity resolution
Transition to HLA specific LIS
HistoTrac (SystemLink)

8. UNEXPECTED PROBLEMS

9. Inclusion of Known HLA typed sample
Newly trained technologist perform NGS library preparation
Continues process and load MiSeq for sequencing without apparent issue
All sequencing metrics are within our stated limits
During data analysis, 1st reviewer notices internal control HLA typing doesn’t match
Investigation determined that the technologist inadvertently flipped the 96-well plate
Entire run was repeated from PCR

10. Inclusion of PhiX sequencing control
Technologist perform NGS library preparation without incident
Loaded libraries on MiSeq

11. Inclusion of PhiX sequencing control
Technologist perform NGS library preparation without incident
Loaded libraries on MiSeq
Low cluster density
Only clusters generated from PhiX
Technologist most likely forgot to denature libraries
Samples repooled, denatured, and sequencing without issue

12. Overclustering Illumina sequencers utilize images for basecalling
Requires space between clusters for optimal accuracy
Repooled libraries using smaller amount

13. Novel Allele
Location of single nucleotide polymorphism (SNP) is important
Core exons & other exons
Intron regions
Splice acceptor and donor sites
Conserved nucleotides sequences required for proper splicing of intron-exon boundaries
~15-50% of human disease caused by splicing mutations
Determines reporting of allele
Synonymous mutation: change in DNA that doesn’t change the encoded amino acid
Non-synonymous mutation: change in DNA that alters amino acid sequence

14. Novel Null Allele Male renal transplant patient SNP: exon 3 bpT157G
Codon: TAT (Y)  TAG (STOP)

15. Novel HLA-A allele Male HSCT workup patient Exon 1 SNP bpA310G
Changes amino acid (AA) from XX to XX
AA changed = new HLA allele
However since AA change outside antigen-recognition domain (exons 2 & 3) the new HLA allele will be in HLA-A*02:01:01G

16. Summary Choice of NGS reagents and platform are instiutional specific
Minor differences between commercial vendors
You will experience a variety of issues with implementation
Important to understand your assay and how each step works
Know how you will handle novel alleles