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.

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.

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.

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.

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.

Low-level parental somatic mosaic SNVs in exomes from a large cohort of trios with diverse suspected Mendelian conditions.

PURPOSE: The goal of this study was to assess the scale of low-level parental mosaicism in exome sequencing (ES) databases. METHODS: We analyzed approximately 2000 family trio ES data sets from the Baylor-Hopkins Center for Mendelian Genomics (BHCMG) and Baylor Genetics (BG). Among apparent de novo single-nucleotide variants identified in the affected probands, we selected rare unique variants with variant allele fraction (VAF) between 30% and 70% in the probands and lower than 10% in one of the parents. RESULTS: Of 102 candidate mosaic variants validated using amplicon-based next-generation sequencing, droplet digital polymerase chain reaction, or blocker displacement amplification, 27 (26.4%) were confirmed to be low- (VAF between 1% and 10%) or very low (VAF <1%) level mosaic. Detection precision in parental samples with two or more alternate reads was 63.6% (BHCMG) and 43.6% (BG). In nine investigated individuals, we observed variability of mosaic ratios among blood, saliva, fibroblast, buccal, hair, and urine samples. CONCLUSION: Our computational pipeline enables robust discrimination between true and false positive candidate mosaic variants and efficient detection of low-level mosaicism in ES samples. We confirm that the presence of two or more alternate reads in the parental sample is a reliable predictor of low-level parental somatic mosaicism.

Nucleic Acid Quantitation with Log-Linear Response Hybridization Probe Sets.

Concentrations of different nucleic acid species in biological samples span many orders of magnitude. A real-time polymerase chain reaction maps the concentration of a target nucleic acid sequence log-linearly into cycle threshold to enable quantitation with a wide dynamic range but suffers from enzymatic biases. Here, we present a general design for constructing hybridization probe sets with highly log-linear response curves to enable accurate enzyme-free quantitation across large ranges (more than 6 logs) of target DNA concentrations. The sensitivity of each component probe is accurately adjusted via formulation stoichiometry to reduce the standard error of target quantitation down to 7%. As a proof of concept, we show multiplexed quantitation of three microRNA species in total RNA of the human brain and liver.

Encoding multiple digital DNA signals in a single analog channel.

For many analytic and biomedical applications, the presence of an analyte above or below a critical concentration is more informative for decision making than the actual concentration value. Straightforward analog-to-digital signal conversion does not take full advantage of the precision and dynamic range of modern sensors. Here, we present and experimentally demonstrate an analog-to-multiple-digital signal conversion, reporting digital signals that indicate whether the concentrations of specific DNA sequences exceed respective threshold values. These threshold values can be individually programmed for each target sequence. Experimentally, we showed representation of four DNA targets' information in a single fluorescence channel.

Highly Sensitive Blocker Displacement Amplification and Droplet Digital PCR Reveal Low-Level Parental FOXF1 Somatic Mosaicism in Families with Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins.

Detection of low-level somatic mosaicism [alternate allele fraction (AAF) ≤ 10%] in parents of affected individuals with the apparent de novo pathogenic variants enables more accurate estimate of recurrence risk. To date, only a few systematic analyses of low-level parental somatic mosaicism have been performed. Herein, highly sensitive blocker displacement amplification, droplet digital PCR, quantitative PCR, long-range PCR, and array comparative genomic hybridization were applied in families with alveolar capillary dysplasia with misalignment of pulmonary veins. We screened 18 unrelated families with the FOXF1 variant previously determined to be apparent de novo (n = 14), of unknown parental origin (n = 1), or inherited from a parent suspected to be somatic and/or germline mosaic (n = 3). We identified four (22%) families with FOXF1 parental somatic mosaic single-nucleotide variants (n = 3) and copy number variant deletion (n = 1) detected in parental blood samples and an AAF ranging between 0.03% and 19%. In one family, mosaic allele ratio in tissues originating from three germ layers ranged between <0.03% and 0.65%. Because the ratio of parental somatic mosaicism have significant implications for the recurrence risk, this study further implies the importance of a systematic screening of parental samples for low-level and very-low-level (AAF ≤ 1%) somatic mosaicism using methods that are more sensitive than those routinely applied in diagnostics.

A recurrent 8 bp frameshifting indel in FOXF1 defines a novel mutation hotspot associated with alveolar capillary dysplasia with misalignment of pulmonary veins.

Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a rare lethal lung developmental disease. Affected infants manifest with severe respiratory distress and refractory pulmonary hypertension and uniformly die in the first month of life. Heterozygous point mutations or copy-number variant deletions involving FOXF1 and/or its upstream lung-specific enhancer on 16q24.1 have been identified in the vast majority of ACDMPV patients. We have previously described two unrelated families with a de novo pathogenic frameshift variant c.691_698del (p.Ala231Argfs*61) in the exon 1 of FOXF1. Here, we present a third unrelated ACDMPV family with the same de novo variant and propose that a direct tandem repeat of eight consecutive nucleotides GCGGCGGC within the ~4 kb CpG island in FOXF1 exon 1 is a novel mutation hotspot causative for ACDMPV.

Predicting DNA hybridization kinetics from sequence.

Hybridization is a key molecular process in biology and biotechnology, but so far there is no predictive model for accurately determining hybridization rate constants based on sequence information. Here, we report a weighted neighbour voting (WNV) prediction algorithm, in which the hybridization rate constant of an unknown sequence is predicted based on similarity reactions with known rate constants. To construct this algorithm we first performed 210 fluorescence kinetics experiments to observe the hybridization kinetics of 100 different DNA target and probe pairs (36 nt sub-sequences of the CYCS and VEGF genes) at temperatures ranging from 28 to 55 °C. Automated feature selection and weighting optimization resulted in a final six-feature WNV model, which can predict hybridization rate constants of new sequences to within a factor of 3 with ∼91% accuracy, based on leave-one-out cross-validation. Accurate prediction of hybridization kinetics allows the design of efficient probe sequences for genomics research.

Multiplexed enrichment of rare DNA variants via sequence-selective and temperature-robust amplification.

Rare DNA-sequence variants hold important clinical and biological information, but existing detection techniques are expensive, complex, allele-specific, or don't allow for significant multiplexing. Here, we report a temperature-robust polymerase-chain-reaction method, which we term blocker displacement amplification (BDA), that selectively amplifies all sequence variants, including single-nucleotide variants (SNVs), within a roughly 20-nucleotide window by 1,000-fold over wild-type sequences. This allows for easy detection and quantitation of hundreds of potential variants originally at ≤0.1% in allele frequency. BDA is compatible with inexpensive thermocycler instrumentation and employs a rationally designed competitive hybridization reaction to achieve comparable enrichment performance across annealing temperatures ranging from 56 °C to 64 °C. To show the sequence generality of BDA, we demonstrate enrichment of 156 SNVs and the reliable detection of single-digit copies. We also show that the BDA detection of rare driver mutations in cell-free DNA samples extracted from the blood plasma of lung-cancer patients is highly consistent with deep sequencing using molecular lineage tags, with a receiver operator characteristic accuracy of 95%.

Publisher Correction: Multiplexed enrichment of rare DNA variants via sequence-selective and temperature-robust amplification.

In the version of this Article originally published, owing to a technical error, the Life Sciences Reporting Summary was not included; this summary is now available.

Continuously tunable nucleic acid hybridization probes.

In silico-designed nucleic acid probes and primers often do not achieve favorable specificity and sensitivity tradeoffs on the first try, and iterative empirical sequence-based optimization is needed, particularly in multiplexed assays. We present a novel, on-the-fly method of tuning probe affinity and selectivity by adjusting the stoichiometry of auxiliary species, which allows for independent and decoupled adjustment of the hybridization yield for different probes in multiplexed assays. Using this method, we achieved near-continuous tuning of probe effective free energy. To demonstrate our approach, we enforced uniform capture efficiency of 31 DNA molecules (GC content, 0-100%), maximized the signal difference for 11 pairs of single-nucleotide variants and performed tunable hybrid capture of mRNA from total RNA. Using the Nanostring nCounter platform, we applied stoichiometric tuning to simultaneously adjust yields for a 24-plex assay, and we show multiplexed quantitation of RNA sequences and variants from formalin-fixed, paraffin-embedded samples.

Cadmium intake and systemic exposure in postmenopausal women and age-matched men who smoke cigarettes.

Mean blood cadmium (B-Cd) concentrations are two- to threefold higher in smokers than in nonsmokers. The basis for this phenomenon is not well understood. We conducted a detailed, multifaceted study of cadmium exposure in smokers. Groups were older smokers (62±4 years, n = 25, 20% male) and nonsmokers (62±3 years, n = 16, 31% male). Each subject's cigarettes were machine smoked, generating individually paired measures of inhaled cadmium (I-Cd) versus B-Cd; I-Cd and B-Cd were each evaluated three times, at monthly intervals. Urine cadmium (U-Cd) was analyzed for comparison. In four smokers, a duplicate-diet study was conducted, along with a kinetic study of plasma cadmium versus B-Cd. Female smokers had a mean B-Cd of 1.21ng Cd/ml, with a nearly 10-fold range (0.29-2.74ng Cd/ml); nonsmokers had a lower mean B-Cd, 0.35ng Cd/ml (p < 0.05), and narrower range (0.20-0.61ng Cd/ml). Means and ranges for males were similar. Estimates of cadmium amounts inhaled daily for our subjects smoking ≥ 20 cigarettes/day were far less than the 15 µg Cd reported to be ingested daily via diet. This I-Cd amount was too low to alone explain the 3.5-fold elevation of B-Cd in our smokers, even assuming greater cadmium absorption via lungs than gastrointestinal tract; cadmium accumulated in smokers' lungs may provide the added cadmium. Finally, B-Cd appeared to be linearly related to I-Cd values in 75% of smokers, whereas 25% had far higher B-Cd, implying a possible heterogeneity among smokers regarding circulating cadmium concentrations and potentially cadmium toxicity.

The impact of ovulation on fallopian tube epithelial cells: evaluating three hypotheses connecting ovulation and serous ovarian cancer.

Ovarian cancer is the most lethal gynecological malignancy affecting American women. Current hypotheses concerning the etiology of ovarian cancer propose that a reduction in the lifetime number of ovulations decreases ovarian cancer risk. Advanced serous carcinoma shares several biomarkers with fallopian tube epithelial cells, suggesting that some forms of ovarian carcinoma may originate in the fallopian tube. Currently, the impact of ovulation on the tubal epithelium is unknown. In CD1 mice, ovulation did not increase tubal epithelial cell (TEC) proliferation as measured by bromodeoxyuridine incorporation and proliferating cell nuclear antigen staining as compared to unstimulated animals. In superovulated mice, an increase in the number of pro-inflammatory macrophages was detected in the oviduct. Ovulation also increased levels of phospho-γH2A.X in TEC, indicating that these cells were susceptible to double-strand DNA breakage following ovulation. To determine which components of ovulation contributed to DNA damage in the fallopian tube, an immortalized baboon TEC cell line and a three-dimensional organ culture system for mouse oviduct and baboon fallopian tubes were developed. TEC did not proliferate or display increased DNA damage in response to the gonadotropins or estradiol alone in vitro. Oxidative stress generated by treatment with hydrogen peroxide or macrophage-conditioned medium increased DNA damage in TEC in culture. Ovulation may impact the fallopian tube epithelium by generating DNA damage and stimulating macrophage infiltration but does not increase proliferation through gonadotropin signaling.