Reverse Complement PCR: fast, low cost amplicon based NGS Chris Mattocks and Daniel Ward, Wessex Regional Genetics Laboratory Festival of Genomics, 31st January 2018

Introduction 2013 WRGL Strategic development plan to rationalise as many laboratory tests onto NGS platform. Two pipelines: Gene panel analysis >> Illumina Nextera based panels Amplicon sequencing for targeted genotyping (constitutional and somatic). Targeted genotyping methodology of choice was 2 step PCR 1° Region of interest PCR 2° sample index PCR.

2 step PCR for generating indexed amplicons (Illumina format) Target specific primers (universal sequence in red) Target DNA 1°PCR product Illumina Indexing primers Sequencing construct ROI P5 P7 Index Seq TS1 TS2 2°PCR

!!! STOP PROCESS !!! 2 step PCR performance Assay 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 Tests setup A B C D E F G Sample !!! STOP PROCESS !!! All data analysed Results obtained

Universal template, universal primer… Cross-contamination of 1°PCR product Cross-contamination of index primers Not easy to monitor using NTCs (i.e not necessarily detectable or informative) Builds over time

Alternative Approaches Ideally: Low cost, single closed tube, early indexing, flexible. 4 primer PCR – not functional in majority of assays. Indexed target specific primers – loss of indexing flexibility and high cost. Post PCR library prep – multi step process, high cost, late indexing, loss of flexibility. In reaction indexed target specific primer synthesis – RC-PCR.

1 step RC-PCR for generating indexed amplicons (Illumina® format) RC probes. 5’ 3’ 5’ 5’ 3’ 3’ 3’ Illumina Indexing kit primers 5’ Target DNA ROI P5 P7 Index Seq GS1 GS2 Sequencing construct

RC-PCR performance Initial optimisation of reaction – probe to primer ratio titration Designs submitted (IDT) for all departmental assays Good performance across all assays without any further optimisation

Applications Specific genotyping tests Variant confirmation Gap infills Rapid low cost single gene screens Copy number analysis? Sample ID testing Methylation analysis 16s RNA

RC-PCR summary features. FAST: PCR > Mix > Clean > Load CHEAP: reagents for 1 PCR, minimal hands on time SECURE: Single, closed tubed indexing and amplification ROBUST: >200 assays, >30,000 clinical reactions Clinically validated for the analysis of both constitutional and somatically acquired variants. Reaction dynamics improve target specificity / reduce primer dimerization potential. Proven ability to perform multiplex RC-PCR. Platform agnostic RC-PCR is IP protected

STRICTLY CONFIDENTIAL

Modelled RC-PCR dynamics Target specific primer synthesis is more aligned with target availability. Reduced potential for: Primer Dimerisation Off target primer binding