Microsatellite Instability Detection in Cancer: A Multiplex qPCR Approach that Obviates the Need for Matching Normal Samples.

BACKGROUND: Microsatellite instability (MSI) indicates DNA mismatch repair deficiency in certain types of cancer, such as colorectal cancer. The current gold standard technique, PCR-capillary electrophoresis (CE), requires matching normal samples and specialized instrumentation. We developed VarTrace, a rapid and low-cost quantitative PCR (qPCR) assay, to evaluate MSI using solely the tumor sample DNA, obviating the requirement for matching normal samples. METHODS: One hundred and one formalin-fixed paraffin-embedded (FFPE) tumor samples were tested using VarTrace and compared with the Promega OncoMate assay utilizing PCR-CE. Tumor percentage limit of detection was evaluated on contrived samples derived from clinical high MSI (MSI-H) samples. Analytical sensitivity, specificity, limit of detection, and input requirements were assessed using synthetic commercial reference standards. RESULTS: VarTrace successfully analyzed all 101 clinical FFPE samples, demonstrating 100% sensitivity and 98% specificity compared to OncoMate. It detected MSI-H with 97% accuracy down to 10% tumor. Analytical studies using synthetic samples showed a limit of detection of 5% variant allele frequency and a limit of input of 0.5 ng. CONCLUSIONS: This study validates VarTrace as a swift, accurate, and economical assay for MSI detection in samples with low tumor percentages without the need for matching normal DNA. VarTrace's capacity for highly sensitive MSI analysis holds potential for enhancing the efficiency of clinical work flows and broadening the availability of this test.

Extended Enrichment for Ultrasensitive Detection of Low-Frequency Mutations by Long Blocker Displacement Amplification.

Detecting low-frequency DNA mutations hotspots cluster is critical for cancer diagnosis but remains challenging. Quantitative PCR (qPCR) is constrained by sensitivity, and allele-specific PCR is restricted by throughput. Here we develop a long blocker displacement amplification (LBDA) coupled with qPCR for ultrasensitive and multiplexed variants detection. By designing long blocker oligos to perfectly match wildtype sequences while mispairing with mutants, long blockers enable 14-44 nt enrichment regions which is 2-fold longer than normal BDA in the experiments. For wild template with a specific nucleotide, LBDA can detect different mutation types down to 0.5 % variant allele frequency (VAF) in one reaction, with median enrichment fold of 1,000 on 21 mutant DNA templates compared to the wild type. We applied LBDA-qPCR to detect KRAS and NRAS mutation hotspots, utilizing a single plex assay capable of covering 81 mutations and tested in synthetic templates and colorectal cancer tissue samples. Moreover, the mutation types were verified through Sanger sequencing, demonstrating concordance with results obtained from next generation sequencing. Overall, LBDA-qPCR provides a simple yet ultrasensitive approach for multiplexed detection of low VAF mutations hotspots, presenting a powerful tool for cancer diagnosis and monitoring.

Rapid, tunable, and multiplexed detection of RNA using convective array PCR.

Detection of RNA targets is typically achieved through RT-qPCR or RNAseq. RT-qPCR is rapid but limited in number and complexity of targets detected, while RNAseq is high-throughput but takes multiple days. We demonstrate simultaneous amplification and detection of 28 distinct RNA targets from a single unsplit purified RNA sample in under 40 minutes using our convective array PCR (caPCR) technology. We integrate tunable strand displacement probes into caPCR to allow detection of RNA species with programmable sequence selectivity for either a single, perfectly matched target sequence or for targets with up to 2 single-nucleotide variants within the probe-binding regions. Tunable probes allow for robust detection of desired RNA species against high homology background sequences and robust detection of RNA species with significant sequence diversity due to community-acquired mutations. As a proof-of-concept, we experimentally demonstrated detection of 7 human coronaviruses and 7 key variants of concern of SARS-CoV-2 in a single assay.

Screening hydrogels for antifibrotic properties by implanting cellularly barcoded alginates in mice and a non-human primate.

Screening implantable biomaterials for antifibrotic properties is constrained by the need for in vivo testing. Here we show that the throughput of in vivo screening can be increased by cellularly barcoding a chemically modified combinatorial library of hydrogel formulations. The method involves the implantation of a mixture of alginate formulations, each barcoded with human umbilical vein endothelial cells from different donors, and the association of the identity and performance of each formulation by genotyping single nucleotide polymorphisms of the cells via next-generation sequencing. We used the method to screen 20 alginate formulations in a single mouse and 100 alginate formulations in a single non-human primate, and identified three lead hydrogel formulations with antifibrotic properties. Encapsulating human islets with one of the formulations led to long-term glycaemic control in a mouse model of diabetes, and coating medical-grade catheters with the other two formulations prevented fibrotic overgrowth. High-throughput screening of barcoded biomaterials in vivo may help identify formulations that enhance the long-term performance of medical devices and of biomaterial-encapsulated therapeutic cells.

Highly Sensitive Blocker Displacement Amplification for Detection of Low-Level JAK2V617F Variant.

BACKGROUND: Key criteria in the diagnostic workup and risk stratification for myeloproliferative neoplasms (MPN) include molecular testing for JAK2V617F and other mutant alleles. Multiple methods for quantitatively detecting nucleotide sequence changes exist, but the lower limit of detection can limit identification of the low-level allele fraction of a variant. We evaluated a recently developed blocker displacement amplification (BDA)-based quantitative PCR platform for detection and quantitation of JAK2V617F variant allele fraction (VAF). METHODS: Clinical samples were tested using BDA, next-generation sequencing (NGS), and droplet digital PCR (ddPCR) in a head-to-head comparison of sensitivity and specificity in detecting the JAK2V617F variant. In total, 112 human genomic DNA specimens previously tested for JAK2V617F gene mutation status with NGS were analyzed, including 12 samples with low-level variants with VAF ≤2%, 6 samples with VAF >2%, and 94 samples with no variant previously identified by NGS. RESULTS: BDA and ddPCR results correlated well across a range of VAFs, with both methods identifying the JAK2V617F variant down to at least 0.05% VAF. NGS of routine sequencing depth was less sensitive, identifying JAK2V617F only at 0.6% VAF. CONCLUSIONS: BDA can provide a cost-effective alternative means to identify low-level variants using instrumentation commonly found in laboratories.

Direct capture and sequencing reveal ultra-short single-stranded DNA in biofluids.

Cell-free DNA (cfDNA) has become the predominant analyte of liquid biopsy; however, recent studies suggest the presence of subnucleosomal-sized DNA fragments in circulation that are likely single-stranded. Here, we report a method called direct capture and sequencing (DCS) tailored to recover such fragments from biofluids by directly capturing them using short degenerate probes followed by single strand-based library preparation and next-generation sequencing. DCS revealed a new DNA population in biofluids, named ultrashort single-stranded DNA (ussDNA). Evaluation of the size distribution and abundance of ussDNA manifested generality of its presence in humans, animal species, and plants. In humans, red blood cells were found to contain abundant ussDNA; plasma-derived ussDNA exhibited modal size at 50 nt. This work reports the presence of an understudied DNA population in circulation, and yet more work is awaiting to study its generation mechanism, tissue of origin, disease implications, etc.

Hairpin Structure Facilitates Multiplex High-Fidelity DNA Amplification in Real-Time Polymerase Chain Reaction.

Clinically and biologically, it is essential to detect rare DNA-sequence variants for early cancer diagnosis or drug-resistance mutation identification. Some of the common quantitative polymerase chain reaction (qPCR)-based variant detection methods are restricted in the limit of detection (LoD) because the DNA polymerases used for these methods have a high polymerase misincorporation rate; thus, the detection sensitivity is sometimes unsatisfactory. With the proofreading activity, high-fidelity (HiFi) DNA polymerases have a 50- to 250-fold higher fidelity. However, there are currently no proper probe-based designs functioning as the fluorescence indicator allowing multiplexed HiFi qPCR reactions, thus restricting the application of HiFi DNA polymerases like the variant detection. We presented the occlusion system, composed of a 5'-overhanged primer with a fluorophore modification and a probe with a short-stem hairpin and a 3' quencher modification. We demonstrated that the occlusion system allowed multiplexing HiFi qPCR reaction, and it was compatible with the current variant-enrichment method to improve the LoD up to 10-fold. Thus, the occlusion system satisfactorily functioned as an efficient fluorescence indicator in HiFi qPCR reactions and allowed the application of HiFi DNA polymerases in variant detection methods to improve detection sensitivity.

Ensemble of nucleic acid absolute quantitation modules for copy number variation detection and RNA profiling.

Current gold standard for absolute quantitation of a specific DNA sequence is droplet digital PCR (ddPCR), which has been applied to copy number variation (CNV) detection. However, the number of quantitation modules in ddPCR is limited by fluorescence channels, which thus limits the CNV sensitivity due to sampling error following Poisson distribution. Here we develop a PCR-based molecular barcoding NGS approach, quantitative amplicon sequencing (QASeq), for accurate absolute quantitation scalable to over 200 quantitation modules. By attaching barcodes to individual target molecules with high efficiency, 2-plex QASeq exhibits higher and more consistent conversion yield than ddPCR in absolute molecule count quantitation. Multiplexed QASeq improves CNV sensitivity allowing confident distinguishment of 2.05 ploidy from normal 2.00 ploidy. We apply multiplexed QASeq to serial longitudinal plasma cfDNA samples from patients with metastatic ERBB2+ (HER2+ ) breast cancer seeking association with tumor progression. We further show an RNA QASeq panel for targeted expression profiling.

Cost-Efficient Sequence-Based Nonextensible Oligonucleotide in Real-Time PCR and High-Throughput Sequencing.

Molecular detection of disease-associated mutations, especially those with low abundance, is essential for academic research and clinical diagnosis. Certain variant detection methods reach satisfactory sensitivity and specificity in detecting rare mutations based on the introduction of blocking oligos to prevent the amplification of wild-type or unwanted templates, thus selectively amplifying and enriching the mutations. These blocking oligos usually suppress PCR amplification through the 3' chemical modifications, with high price, slow synthesis, and reduced purity. Herein, we introduce chemistry-free designs to block enzymatic extension during PCR by the steric hindrance from the secondary structures attached to the 3' end of the oligos (nonextensible oligonucleotide, NEO). We demonstrated that NEO efficiently prohibited the extension of both Taq and high-fidelity DNA polymerases. By further applying NEO as blockers in blocker displacement amplification (BDA) qPCR, multiplex BDA (mBDA) NGS, and quantitative BDA (QBDA) NGS methods, we showed that NEO blockers had performance comparable with previously validated chemical modifications. Comparison experiments using QBDA with NEO blockers and droplet digital PCR (ddPCR) on clinical formalin-fixed paraffin-embedded (FFPE) samples exhibited 100% concordance. Lastly, the ability of NEO to adjust plex uniformity through changes of PCR amplification efficiency was demonstrated in an 80-plex NGS panel.

Designing highly multiplex PCR primer sets with Simulated Annealing Design using Dimer Likelihood Estimation (SADDLE).

One major challenge in the design of highly multiplexed PCR primer sets is the large number of potential primer dimer species that grows quadratically with the number of primers to be designed. Simultaneously, there are exponentially many choices for multiplex primer sequence selection, resulting in systematic evaluation approaches being computationally intractable. Here, we present and experimentally validate Simulated Annealing Design using Dimer Likelihood Estimation (SADDLE), a stochastic algorithm for design of multiplex PCR primer sets that minimize primer dimer formation. In a 96-plex PCR primer set (192 primers), the fraction of primer dimers decreases from 90.7% in a naively designed primer set to 4.9% in our optimized primer set. Even when scaling to 384-plex (768 primers), the optimized primer set maintains low dimer fraction. In addition to NGS, SADDLE-designed primer sets can also be used in qPCR settings to allow highly multiplexed detection of gene fusions in cDNA, with a single-tube assay comprising 60 primers detecting 56 distinct gene fusions recurrently observed in lung cancer.

Limitations and opportunities of technologies for the analysis of cell-free DNA in cancer diagnostics.

Cell-free DNA (cfDNA) in the circulating blood plasma of patients with cancer contains tumour-derived DNA sequences that can serve as biomarkers for guiding therapy, for the monitoring of drug resistance, and for the early detection of cancers. However, the analysis of cfDNA for clinical diagnostic applications remains challenging because of the low concentrations of cfDNA, and because cfDNA is fragmented into short lengths and is susceptible to chemical damage. Barcodes of unique molecular identifiers have been implemented to overcome the intrinsic errors of next-generation sequencing, which is the prevailing method for highly multiplexed cfDNA analysis. However, a number of methodological and pre-analytical factors limit the clinical sensitivity of the cfDNA-based detection of cancers from liquid biopsies. In this Review, we describe the state-of-the-art technologies for cfDNA analysis, with emphasis on multiplexing strategies, and discuss outstanding biological and technical challenges that, if addressed, would substantially improve cancer diagnostics and patient care.

Single-Tube qPCR Detection and Quantitation of Hotspot Mutations Down to 0.01% Variant Allele Fraction.

Clinically and biologically, rare DNA sequence variants are significant and informative. However, existing common detection technologies are either complex and time-consuming 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 single nucleotide variant or small insertions and deletions (INDELs) 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 on peripheral blood mononuclear cell clinical samples showed 100% concordance for samples with mutations at ≥ 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.

Calibration-free NGS quantitation of mutations below 0.01% VAF.

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 errors for rare mutation detection, the sequencing depth requirement is high. Here, we present Quantitative Blocker Displacement Amplification (QBDA) which integrates sequence-selective variant enrichment into UMI quantitation for accurate quantitation of mutations below 0.01% VAF at only 23,000X 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 0.001% VAF at a single locus with less than 4 million sequencing reads, allowing sensitive MRD detection in patients during complete remission. In a pan-cancer panel and a melanoma hotspot panel, we detect mutations down to 0.1% VAF using only 1 million reads. QBDA provides a convenient and versatile method for sensitive mutation quantitation using low-depth sequencing.

Oncogene Concatenated Enriched Amplicon Nanopore Sequencing for rapid, accurate, and affordable somatic mutation detection.

We develop the Oncogene Concatenated Enriched Amplicon Nanopore Sequencing (OCEANS) method, in which variants with low variant allele frequency (VAFs) are amplified and subsequently concatenated for Nanopore Sequencing. OCEANS allows accurate detection of somatic mutations with VAF limits of detection between 0.05 and 1%. We construct 4 distinct multi-gene OCEANS panels targeting recurrent mutations in acute myeloid leukemia, melanoma, non-small- cell lung cancer, and hepatocellular carcinoma and validate them on clinical samples. By demonstrating detection of low VAF single nucleotide variant mutations using Nanopore Sequencing, OCEANS is poised to enable same-day clinical sequencing panels.

Highly multiplexed rapid DNA detection with single-nucleotide specificity via convective PCR in a portable device.

Assays for the molecular detection of nucleic acids are typically constrained by the level of multiplexing (this is the case for the quantitative polymerase chain reaction (qPCR) and for isothermal amplification), turnaround times (as with microarrays and next-generation sequencing), quantification accuracy (isothermal amplification, microarrays and nanopore sequencing) or specificity for single-nucleotide differences (microarrays and nanopore sequencing). Here we show that a portable and battery-powered PCR assay performed in a toroidal convection chamber housing a microarray of fluorescently quenched oligonucleotide probes allows for the rapid and sensitive quantification of multiple DNA targets with single-nucleotide discrimination. The assay offers a limit of detection of 10 DNA copies within 30 min of turnaround time and a dynamic range spanning 4 orders of magnitude of DNA concentration, and we show its performance by detecting 20 genomic loci and 30 single-nucleotide polymorphisms in human genomic DNA samples, and 15 bacterial species in clinical isolates. Portable devices for the fast and highly multiplexed detection of nucleic acids may offer advantages in point-of-care diagnostics.

Predicting stability of DNA bulge at mononucleotide microsatellite.

Mononucleotide microsatellites are clinically and forensically crucial DNA sequences due to their high mutability and abundance in the human genome. As a mutagenic intermediate of an indel in a microsatellite and a consequence of probe hybridization after such mutagenesis, a bulge with structural degeneracy sliding within a microsatellite is formed. Stability of such dynamic bulges, however, is still poorly understood despite their critical role in cancer genomics and neurological disease studies. In this paper, we have built a model that predicts the thermodynamics of a sliding bulge at a microsatellite. We first identified 40 common bulge states that can be assembled into any sliding bulges, and then characterized them with toehold exchange energy measurement and the partition function. Our model, which is the first to predict the free energy of sliding bulges with more than three repeats, can infer the stability penalty of a sliding bulge of any sequence and length with a median prediction error of 0.22 kcal/mol. Patterns from the prediction clearly explain landscapes of microsatellites observed in the literature, such as higher mutation rates of longer microsatellites and C/G microsatellites.

A deep learning model for predicting next-generation sequencing depth from DNA sequence.

Targeted high-throughput DNA sequencing is a primary approach for genomics and molecular diagnostics, and more recently as a readout for DNA information storage. Oligonucleotide probes used to enrich gene loci of interest have different hybridization kinetics, resulting in non-uniform coverage that increases sequencing costs and decreases sequencing sensitivities. Here, we present a deep learning model (DLM) for predicting Next-Generation Sequencing (NGS) depth from DNA probe sequences. Our DLM includes a bidirectional recurrent neural network that takes as input both DNA nucleotide identities as well as the calculated probability of the nucleotide being unpaired. We apply our DLM to three different NGS panels: a 39,145-plex panel for human single nucleotide polymorphisms (SNP), a 2000-plex panel for human long non-coding RNA (lncRNA), and a 7373-plex panel targeting non-human sequences for DNA information storage. In cross-validation, our DLM predicts sequencing depth to within a factor of 3 with 93% accuracy for the SNP panel, and 99% accuracy for the non-human panel. In independent testing, the DLM predicts the lncRNA panel with 89% accuracy when trained on the SNP panel. The same model is also effective at predicting the measured single-plex kinetic rate constants of DNA hybridization and strand displacement.

Confirming putative variants at ≤ 5% allele frequency using allele enrichment and Sanger sequencing.

Whole exome sequencing (WES) is used to identify mutations in a patient's tumor DNA that are predictive of tumor behavior, including the likelihood of response or resistance to cancer therapy. WES has a mutation limit of detection (LoD) at variant allele frequencies (VAF) of 5%. Putative mutations called at ≤ 5% VAF are frequently due to sequencing errors, therefore reporting these subclonal mutations incurs risk of significant false positives. Here we performed ~ 1000 × WES on fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissue biopsy samples from a non-small cell lung cancer patient, and identified 226 putative mutations at between 0.5 and 5% VAF. Each variant was then tested using NuProbe NGSure, to confirm the original WES calls. NGSure utilizes Blocker Displacement Amplification to first enrich the allelic fraction of the mutation and then uses Sanger sequencing to determine mutation identity. Results showed that 52% of the 226 (117) putative variants were disconfirmed, among which 2% (5) putative variants were found to be misidentified in WES. In the 66 cancer-related variants, the disconfirmed rate was 82% (54/66). This data demonstrates Blocker Displacement Amplification allelic enrichment coupled with Sanger sequencing can be used to confirm putative mutations ≤ 5% VAF. By implementing this method, next-generation sequencing can reliably report low-level variants at a high sensitivity, without the cost of high sequencing depth.

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

DNA sequence variants with allele fractions below 1% are difficult to detect and quantify by sequencing owing to intrinsic errors in sequencing-by-synthesis methods. Although molecular-identifier barcodes can detect mutations with a variant-allele frequency (VAF) as low as 0.1% using next-generation sequencing (NGS), sequencing depths of over 25,000× are required, thus hampering the detection of mutations at high sensitivity in patient samples and in most samples used in research. Here we show that low-frequency DNA variants can be detected via low-depth multiplexed NGS after their amplification, by a median of 300-fold, using polymerase chain reaction and rationally designed 'blocker' oligonucleotides that bind to the variants. Using an 80-plex NGS panel and a sequencing depth of 250×, we detected single nucleotide polymorphisms with a VAF of 0.019% and contamination in human cell lines at a VAF as low as 0.07%. With a 16-plex NGS panel covering 145 mutations across 9 genes involved in melanoma, we detected low-VAF mutations (0.2-5%) in 7 out of the 19 samples of freshly frozen tumour biopsies, suggesting that tumour heterogeneity could be notably higher than previously recognized.

High sensitivity sanger sequencing detection of BRAF mutations in metastatic melanoma FFPE tissue specimens.

Mutations in the BRAF gene at or near the p. V600 locus are informative for therapy selection, but current methods for analyzing FFPE tissue DNA generally have a limit of detection of 5% variant allele frequency (VAF), or are limited to the single variant (V600E). These can result in false negatives for samples with low VAFs due to low tumor content or subclonal heterogeneity, or harbor non-V600 mutations. Here, we show that Sanger sequencing using the NuProbe VarTrace BRAF assay, based on the Blocker Displacement Amplification (BDA) technology, is capable of detecting BRAF V600 mutations down to 0.20% VAF from FFPE lymph node tissue samples. Comparison experiments on adjacent tissue sections using BDA Sanger, immunohistochemistry (IHC), digital droplet PCR (ddPCR), and NGS showed 100% concordance among all 4 methods for samples with BRAF mutations at ≥ 1% VAF, though ddPCR did not distinguish the V600K mutation from the V600E mutation. BDA Sanger, ddPCR, and NGS (with orthogonal confirmation) were also pairwise concordant for lower VAF mutations down to 0.26% VAF, but IHC produced a false negative. Thus, we have shown that Sanger sequencing can be effective for rapid detection and quantitation of multiple low VAF BRAF mutations from FFPE samples. BDA Sanger method also enabled detection and quantitation of less frequent, potentially actionable non-V600 mutations as demonstrated by synthetic samples.