Selective multiplexed enrichment for the detection and quantitation of low-fraction DNA variants via low-depth sequencing

Song, Ping; Chen, Sherry X.; Yan, Yan Helen; Pinto, Alessandro; Cheng, Lauren Y.; Dai, Peng; Patel, Abhijit A.; Zhang, David Y.

doi:10.1038/s41551-021-00713-0

Cited by 37 publications

(34 citation statements)

References 48 publications

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“…What is more, the Occlusion System could provide an easy, ready-to-use design solution to any panels with universal or specific 5' overhang sequences during the PCR step. We validated our hypothesis by applying the Occlusion System to two previously designed NGS panels, one is a 80-plex panel 46 ; the other is 179-plex 49 . In the presence of the Occlusion Probe, the percentage of reads of 80-plex target amplicons increased by 100% (from 40% to 80%), the 179-plex target reads increased by 700% (Figure 4C).…”

Section: Improvement Of Pcr Efficiency -Reduction Of Primer Dimer Formation By the Occlusion Systemmentioning

confidence: 82%

“…The current multiplex PCR method AmpliSeq 45 is primarily composed of enzymatic digestion of excessive primer sequences post amplicon-generation PCR, followed by a ligation step of adaptor sequences. Other methods reported 44,46 used a second PCR step to append adaptor sequence on the amplicons, which saved hands-on time with higher efficiency as the ligation efficiency is usually less than 30%. However, both methods sophisticate the total sequencing workflow.…”

Section: Introductionmentioning

confidence: 99%

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Hairpin structure facilitates multiplex high-fidelity DNA amplification in real-time PCR

Zhang

Pinto²,

Dai

et al. 2021

Preprint

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Effective polymerase chain reactions (PCR) are important in bio-laboratories. It is essential to detect rare DNA-sequence variants for early cancer diagnosis or for drug-resistance mutations identification. Some of the common detection quantitative PCR (qPCR) methods are restricted in the limit of detection (LoD) because of the high polymerase misincorporation rate in Taq DNA polymerases. High-fidelity (HiFi) DNA polymerases have a 50- to 250-fold higher fidelity. Yet, there are currently no proper designs for multiplexed HiFi qPCR reactions. Moreover, the popularity of targeting highly multiplex DNA sequences requires minimizing PCR side products, as the potential of dimerization grows quadratically as the plexes of primers increases. Efforts tried before were either an add-on step, or technology-specific, or requiring high-level computing skills. There lacks an easy-to-apply and cost-effective method for dimerization reduction. Here, we presented the Occlusion System, composed of a 5’-overhanged primer and a probe with a short-stem hairpin. We demonstrated that it allowed multiplexing high-fidelity qPCR reaction, it was also compatible with the current variant-enrichment method to improve the LoD by 10-fold. Further, we found that the Occlusion System reduced the dimerization up to 10-fold in highly multiplexed PCR. Thus, the Occlusion System satisfactorily improved both qPCR sensitivity and PCR efficiency.

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Section: Improvement Of Pcr Efficiency -Reduction Of Primer Dimer Formation By the Occlusion Systemmentioning

confidence: 82%

Section: Introductionmentioning

confidence: 99%

Hairpin structure facilitates multiplex high-fidelity DNA amplification in real-time PCR

Zhang

Pinto²,

Dai

et al. 2021

Preprint

Self Cite

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“…The combination of UMI and high input amount leads to sequencing depth unaffordable for many researchers, clinicians and patients. Blocker displacement amplification (BDA) 24,25 enriches variant alleles by introducing rationally designed blocker oligonucleotides that competes with forward primer to suppress the amplification of wild type molecules. BDA allows detection of rare mutations using low sequencing depth, but loses VAF quantitation without calibration.…”

Section: Introductionmentioning

confidence: 99%

Calibration-free NGS Quantitation of Mutations below 0.01% VAF

Dai

Chen

et al. 2021

Preprint

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The quantitation of rare somatic mutations is essential for basic research and translational clinical applications including minimal residual disease (MRD) detection. Though unique molecular identifier (UMI) has suppressed sequencing error and allowed detection rare mutation, the sequencing depth requirement is high. The blocker displacement amplification (BDA) allele enrichment method allows detection of rare mutations using low sequencing depth, but requires calibration to accurately quantitate the VAF of novel mutations. Here, we present Quantitative Blocker Displacement Amplification (QBDA), a method that allows accurate detection and quantitation of mutations below 0.01% VAF at only 23,000X depth. QBDA integrates sequence-selective variant enrichment into UMI quantitation allowing confident detection of rare mutations and reduced sequencing depth. Using a panel of 20 genes recurrently altered in acute myeloid leukemia, we demonstrate quantitation of various mutations including single base substitutions and indels down to a VAF of 0.001% at a single locus with less than 4 million sequencing reads, allowing a sensitive minimal residual disease (MRD) detection in patients during complete remission. In a comprehensive pan-cancer panel covering 61 genes and a melanoma hotspot panel covering 8 genes, we detect mutations down to 0.1% VAF using only 1 million reads in a broad range of clinical samples including cell-free DNA and FFPE DNA, enabling tissue or liquid biopsy genetic tests with de-centralized sequencing instruments. QBDA thus provides a convenient and versatile method for sensitive mutation quantitation using low-depth sequencing.

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“…However, previous BDA applications in real-time PCR focused on applying TaqMan probes downstream to the blocker enrichment region to indicate the generation of the amplicon, thus non-specific to any mutations that happen within the blocker enrichment region. It requires at least two reactions 17 or additional technology like Sanger sequencing 18 , next-generation sequencing (NGS) 19 or third-generation sequencing 20 , to determine the identity of the enriched variant, increasing the total cost and overall turnaround time. Here we present an allele-specific BDA (As-BDA) method, which takes advantage of the effectiveness of BDA in rare variant enrichment and integrates allele-specific TaqMan probe for single nucleotide variant (SNV) genotyping.…”

Section: Introductionmentioning

confidence: 99%

Single Tube qPCR detection and quantitation of hotspot mutations down to 0.01% VAF

Zhang

Rodríguez²,

Cheng

et al. 2021

Preprint

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Clinically and biologically, rare DNA sequence variants are significant and informative. However, existing detection technologies are either complex in workflow, or restricted in the limit of detection (LoD), or do not allow for multiplexing. Blocker displacement amplification (BDA) method can stably and effectively detect and enrich multiple rare variants with LoD around 0.1% variant allele fraction (VAF). Nonetheless, the detailed mutation information has to be identified by additional sequencing technologies. Here, we present allele-specific BDA (As-BDA), a method combining BDA with allele-specific TaqMan (As-TaqMan) probes for effective variant enrichment and simultaneous SNV profiling. We demonstrated that As-BDA could detect mutations down to 0.01% VAF. Further, As-BDA could detect up to four mutations with low to 0.1% VAF per reaction using only 15 ng DNA input. The median error of As-BDA in VAF determination is approximately 9.1%. Comparison experiments using As-BDA and droplet digital PCR (ddPCR) on peripheral blood mononuclear cell (PBMC) clinical samples showed 100% concordance for samples with mutations at 3 0.1% VAF. Hence, we have shown that As-BDA can achieve simultaneous enrichment and identification of multiple targeted mutations within the same reaction with high clinical sensitivity and specificity, thus helpful for clinical diagnosis.

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Selective multiplexed enrichment for the detection and quantitation of low-fraction DNA variants via low-depth sequencing

Cited by 37 publications

References 48 publications

Hairpin structure facilitates multiplex high-fidelity DNA amplification in real-time PCR

Hairpin structure facilitates multiplex high-fidelity DNA amplification in real-time PCR

Calibration-free NGS Quantitation of Mutations below 0.01% VAF

Single Tube qPCR detection and quantitation of hotspot mutations down to 0.01% VAF

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