1. Multiplexed Elimination of Wild-Type DNA and High-Resolution Melting Prior to Targeted Resequencing of Liquid Biopsies
I. Ladas, M. Fitarelli-Kiehl, C. Song, V.A. Adalsteinsson, H. A. Parsons, N.U. Lin, N. Wagle, G.M. Makrigiorgos
October 2017
© Copyright 2017 by the American Association for Clinical Chemistry

2. Introduction
There is mounting evidence that tumor mutations identified in cell-free DNA (cfDNA) can potentially act as a powerful liquid biopsy-based diagnostic tool
Clinical studies indicate the use of cfDNA to complement or replace tissue biopsies.
The limited amount of cfDNA obtained from a standard blood draw and the excess amount of circulating wild-type (WT) over mutated DNA often confound interpretation of the results.
Here we present a novel, multiplexed mutation enrichment method that can be combined with multiplexed high resolution melting (HRM) to pre-screen samples for mutations prior to next generation sequencing (NGS).

3. Method
We utilize a nuclease, DSN, specific for double stranded DNA together with overlapping probes that bracket the mutation site(s) on both DNA strands of the interrogated double stranded DNA.
At modestly high temperatures (~65oC) the probes bind their targeted sites and the nuclease eliminates WT DNA, thus enriching for mutations on multiple targets simultaneously.
This approach, termed ‘No-Denaturation Nuclease assisted Minor-allele enrichment with Probe-Overlap, ND-NaME-PrO’ is a modification of the original NaME-PrO developed by our group, adapted here to a homogeneous closed-tube reaction.

4. ND-NaME-PrO principle and workflow
A. ND-NaME-PrO modus operandi: at elevated temperatures ~65oC the probes bind their complementary target DNA strands. DSN digests fully matched templates on multiple targets simultaneously, whereas mismatched sequences remain substantially undigested, thereby resulting in mutation enrichment upon subsequent amplification. Note: ND-NaME-PrO requires amplification using short amplicons flanking the mutation site, eg < 150bp.
B. The workflow comprises an initial multiplex amplification from genomic DNA or cfDNA that enriches for the targeted DNA sites. This is followed by multiplexed ND-NaME-PrO reaction and by nested multiplexed anchor-tail PCR. The product is screened via multiplexed PCR-HRM mutation scanning. HRM-positive samples undergo library formation and NGS, while negative samples may not be screened further.

5. Question
Can ND-NaME-PrO enrich simultaneously multiple targets? How many targets maximum can be enriched?

6. Notes
ND-NaME-PrO is initiated by adding DSN enzyme in a PCR tube that includes DNA sample and all other components of the reaction and immediately placed in a PCR machine pre-heated to 65oC.
At 65oC DNA undergoes temporary regional denaturation (DNA “breathing”) and is only partially denatured.
The data are consistent with temporary binding of NaME-PrO probes to their respective target(s) during DNA breathing and enabling DSN to nick DNA at the targeted sites. WT DNA is preferentially digested as compared to mutant DNA.
The combination of enzyme concentration, probe concentration and input DNA concentration during ND-NaME-PrO is critical in order to achieve efficient mutation enrichment.
Off-target DNA nicking for both mutant and WT DNA also takes place. Overall however, DNA segments in the vicinity of the mutations are digested significantly less relative to the corresponding WT segments.

7. Question
Why does the mutation enrichment provided by ND-NaME-PrO decrease when primers for long amplicons encompassing the mutations are used to amplify DSN-digested DNA targets? And why do shorter amplicons provide higher enrichment?

8. Results: Single-plex ND-NaME-PrO applied on PCR products
Single-plex ND-NaME-PrO was applied for KRAS (c.35G>T, p.G12V) on serially decreasing dilutions of mutated KRAS in WT DNA (A). Mutation abundance was assessed via droplet digital PCR performed before and after ND-NaME-PrO. (B). Mutation enrichment obtained for KRAS mutations after ND-NaME-PrO ranged from 10-fold to 120-fold for 1% and 0.01% mutation frequencies respectively. The lower the initial mutation abundance the bigger the enrichment obtained becomes.

9. Results: Multiplexed 10-plex ND-NaME-PrO followed by 10-plex PCR and 10-plex HRM scanning. Experiments shown contain a single mutated target at serially decreasing dilutions.
Targets tested within 10-plex reactions include BRAF (A), IDH1 (B), KRAS (C), and JAK2 (D). In the top 4 panels, samples run in parallel while omitting DSN treatment are shown. In the middle 4 panels, samples treated with ND-NaME-PrO (homogeneous, closed-tube process) are shown. In bottom 4 panels, samples treated with NaME-PrO (non-homogenous process including DNA denaturation) is shown. All mutation samples were assessed in duplicate (2) and WT controls in quadruplicate (4).

10. Results: 10-plex ND-NaME-PrO-HRM followed by MiSeq performed in circulating tumor DNA (ctDNA) containing mutations in 10 different targets.
No-treatment samples without DSN were processed in parallel for comparison (A and D). Multiplexed mutation scanning prescreening performed by multiplexed HRM (B and E). Variant frequency at the 10 mutated targets on cfDNA as derived via targeted resequencing (MiSeq) for no-treatment samples (blue bars) and ND-NaME-treated samples (red bars) (C and F).

11. Results summary
Serial dilution of KRAS mutation-containing DNA yields mutation enrichment by 10- to 120-fold and detection of allelic fractions down to 0.01%, following ND-NaME-PrO.
Multiplexed ND-NaME-PrO combined with multiplexed PCR-HRM enables mutation scanning over 10–20 DNA amplicons simultaneously.
ND-NaME-PrO applied on cfDNA from clinical samples enables mutation enrichment and HRM scanning over 10 DNA targets.
cfDNA mutations in HRM-positive samples were enriched up to approximately 100-fold (average approximately 25-fold) and identified via targeted resequencing.

12. Discussion
Closed-tube homogeneous ND-NaME-PrO combined with multiplexed HRM is a convenient approach to efficiently enrich for mutations on multiple DNA targets and to enable pre-screening before targeted resequencing.
Here, combining mutation enrichment by ND-NaME-PrO with nested multiplexed PCR and HRM resulted in sensitive HRM scanning for mutations for targets simultaneously
This development opens up the possibility of filtering out uninformative samples using HRM and concentrating targeted resequencing efforts and resources to just the mutation-containing samples.

13. Question
Why does multiplexed HRM require mutation enrichment to enable sensitive mutation scanning?