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

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

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

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

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

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

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)

UNEXPECTED PROBLEMS

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

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

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

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

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

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

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

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