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

2. 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.

3. 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

4. !!! STOP PROCESS !!! 2 step PCR performance Assay 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

5. 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

6. 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.

7. 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

8. 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

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

10. 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

11. STRICTLY CONFIDENTIAL

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