Structural Alterations Driving Castration-Resistant Prostate Cancer Revealed by Linked-Read Genome Sequencing.

Nearly all prostate cancer deaths are from metastatic castration-resistant prostate cancer (mCRPC), but there have been few whole-genome sequencing (WGS) studies of this disease state. We performed linked-read WGS on 23 mCRPC biopsy specimens and analyzed cell-free DNA sequencing data from 86 patients with mCRPC. In addition to frequent rearrangements affecting known prostate cancer genes, we observed complex rearrangements of the AR locus in most cases. Unexpectedly, these rearrangements include highly recurrent tandem duplications involving an upstream enhancer of AR in 70%-87% of cases compared with <2% of primary prostate cancers. A subset of cases displayed AR or MYC enhancer duplication in the context of a genome-wide tandem duplicator phenotype associated with CDK12 inactivation. Our findings highlight the complex genomic structure of mCRPC, nominate alterations that may inform prostate cancer treatment, and suggest that additional recurrent events in the non-coding mCRPC genome remain to be discovered.

MAESTRO-Pool Enables Highly Parallel and Specific Mutation-Enrichment Sequencing for Minimal Residual Disease Detection in Cohort Studies.

BACKGROUND: Tracing patient-specific tumor mutations in cell-free DNA (cfDNA) for minimal residual disease (MRD) detection is promising but challenging. Assaying more mutations and cfDNA stands to improve MRD detection but requires highly accurate, efficient sequencing methods and proper calibration to prevent false detection with bespoke tests. METHODS: MAESTRO (Minor Allele Enriched Sequencing Through Recognition Oligonucleotides) uses mutation-specific oligonucleotide probes to enrich cfDNA libraries for tumor mutations and enable their accurate detection with minimal sequencing. A new approach, MAESTRO-Pool, which entails pooling MAESTRO probes for all patients and applying these to all samples from all patients, was used to screen for 22 333 tumor mutations from 9 melanoma patients in 98 plasma samples. This enabled quantification of MRD detection in patient-matched samples and false detection in unmatched samples from other patients. To detect MRD, a new dynamic MRD caller was used that computes a probability for MRD detection based on the number of mutations and cfDNA molecules sequenced, thereby calibrating for variations in each bespoke test. RESULTS: MAESTRO-Pool enabled sensitive detection of MRD down to 0.78 parts per million (ppm), reflecting a 10- to 100-fold improvement over existing tests. Of the 8 MRD positive samples with ultra-low tumor fractions <10 ppm, 7 were either in upward-trend preceding recurrence or downward-trend aligning with response. Of 784 patient-unmatched tests, only one was found as MRD positive (tumor fraction = 2.7 ppm), suggesting high specificity. CONCLUSIONS: MAESTRO-Pool enables massively parallel, tumor-informed MRD testing with concurrent benchmarking of bespoke MRD tests. Meanwhile, our new MRD caller enables more mutations and cfDNA molecules to be tested without compromising specificity. These improve the ability for detecting traces of MRD from blood.

Duplex-Repair enables highly accurate sequencing, despite DNA damage.

Accurate DNA sequencing is crucial in biomedicine. Underlying the most accurate methods is the assumption that a mutation is true if altered bases are present on both strands of the DNA duplex. We now show that this assumption can be wrong. We establish that current methods to prepare DNA for sequencing, via 'End Repair/dA-Tailing,' may substantially resynthesize strands, leading amplifiable lesions or alterations on one strand to become indiscernible from true mutations on both strands. Indeed, we discovered that 7-17% and 32-57% of interior 'duplex base pairs' from cell-free DNA and formalin-fixed tumor biopsies, respectively, could be resynthesized in vitro and potentially introduce false mutations. To address this, we present Duplex-Repair, and show that it limits interior duplex base pair resynthesis by 8- to 464-fold, rescues the impact of induced DNA damage, and affords up to 8.9-fold more accurate duplex sequencing. Our study uncovers a major Achilles' heel in sequencing and offers a solution to restore high accuracy.

NGS-based identification and tracing of microsatellite instability from minute amounts DNA using inter-Alu-PCR.

Sensitive detection of microsatellite instability (MSI) in tissue or liquid biopsies using next generation sequencing (NGS) has growing prognostic and predictive applications in cancer. However, the complexities of NGS make it cumbersome as compared to established multiplex-PCR detection of MSI. We present a new approach to detect MSI using inter-Alu-PCR followed by targeted NGS, that combines the practical advantages of multiplexed-PCR with the breadth of information provided by NGS. Inter-Alu-PCR employs poly-adenine repeats of variable length present in every Alu element and provides a massively-parallel, rapid approach to capture poly-A-rich genomic fractions within short 80-150bp amplicons generated from adjacent Alu-sequences. A custom-made software analysis tool, MSI-tracer, enables Alu-associated MSI detection from tissue biopsies or MSI-tracing at low-levels in circulating-DNA. MSI-associated indels at somatic-indel frequencies of 0.05-1.5% can be detected depending on the availability of matching normal tissue and the extent of instability. Due to the high Alu copy-number in human genomes, a single inter-Alu-PCR retrieves enough information for identification of MSI-associated-indels from ∼100 pg circulating-DNA, reducing current limits by ∼2-orders of magnitude and equivalent to circulating-DNA obtained from finger-sticks. The combined practical and informational advantages of inter-Alu-PCR make it a powerful tool for identifying tissue-MSI-status or tracing MSI-associated-indels in liquid biopsies.

Implementation of a prostate cancer-specific targeted sequencing panel for credentialing of patient-derived cell lines and genomic characterization of patient samples.

BACKGROUND: Primary and metastatic prostate cancers have low mutation rates and recurrent alterations in a small set of genes, enabling targeted sequencing of prostate cancer-associated genes as an efficient approach to characterizing patient samples (compared to whole-exome and whole-genome sequencing). For example, targeted sequencing provides a flexible, rapid, and cost-effective method for genomic assessment of patient-derived cell lines to evaluate fidelity to initial patient tumor samples. METHODS: We developed a prostate cancer-specific targeted next-generation sequencing (NGS) panel to detect alterations in 62 prostate cancer-associated genes as well as recurring gene fusions with ETS family members, representing the majority of common alterations in prostate cancer. We tested this panel on primary prostate cancer tissues and blood biopsies from patients with metastatic prostate cancer. We generated patient-derived cell lines from primary prostate cancers using conditional reprogramming methods and applied targeted sequencing to evaluate the fidelity of these cell lines to the original patient tumors. RESULTS: The prostate cancer-specific panel identified biologically and clinically relevant alterations, including point mutations in driver oncogenes and ETS family fusion genes, in tumor tissues from 29 radical prostatectomy samples. The targeted panel also identified genomic alterations in cell-free DNA and circulating tumor cells (CTCs) from patients with metastatic prostate cancer, and in standard prostate cancer cell lines. We used the targeted panel to sequence our set of patient-derived cell lines; however, no prostate cancer-specific mutations were identified in the tumor-derived cell lines, suggesting preferential outgrowth of normal prostate epithelial cells. CONCLUSIONS: We evaluated a prostate cancer-specific targeted NGS panel to detect common and clinically relevant alterations (including ETS family gene fusions) in prostate cancer. The panel detected driver mutations in a diverse set of clinical samples of prostate cancer, including fresh-frozen tumors, cell-free DNA, CTCs, and cell lines. Targeted sequencing of patient-derived cell lines highlights the challenge of deriving cell lines from primary prostate cancers and the importance of genomic characterization to credential candidate cell lines. Our study supports that a prostate cancer-specific targeted sequencing panel provides an efficient, clinically feasible approach to identify genetic alterations across a spectrum of prostate cancer samples and cell lines.

Recent Developments in Mutation Enrichment and Detection Technologies.

BACKGROUND: Presence of excess unaltered, wild-type DNA (wtDNA) providing information of little clinical value may often mask low-level mutations containing important diagnostic or therapeutic clues. This is a recurring hurdle in biotechnology and medicine, including cancer, prenatal diagnosis, infectious diseases, and organ transplantation. Mutation enrichment techniques that allow reduction of unwanted DNA to enable the detection of low-level mutations have emerged since the early 1990s. They are continuously being refined and updated with new technologies. The burgeoning interest in liquid biopsies for residual cancer monitoring, detection of resistance to therapy, and early cancer detection has driven an expanded interest in new and improved methodologies for practical and effective mutation enrichment and detection of low-level mutations of clinical relevance. CONTENT: Newly developed mutation enrichment technologies are described and grouped according to the main principle of operation, PCR-blocking technologies, enzymatic methods, and physicochemical approaches. Special emphasis is given to technologies enabling pre-PCR blockage of wtDNA to bypass PCR errors [nuclease-assisted minor-allele enrichment assay with overlapping probes (NaME-PrO) and UV-mediated cross-linking minor allele enrichment (UVME)] or providing high multiplexity followed by next-generation sequencing [Minor allele enriched sequencing through recognition oligonucleotides (MAESTRO)]. SUMMARY: This review summarizes technological developments in rare mutation enrichment over the last 12 years, complementing pre-2010 reviews on this topic. The expanding field of liquid biopsy calls for improved limits of detection (LOD) and highly parallel applications, along with the traditional requirements for accuracy, speed, and cost-effectiveness. The current technologies are reviewed with regards to these new requirements.

A nuclease-polymerase chain reaction enables amplification of probes used for capture-based DNA target enrichment.

DNA target enrichment via hybridization capture is a commonly adopted approach which remains expensive due in-part to using biotinylated-probe panels. Here we provide a novel isothermal amplification reaction to amplify rapidly existing probe panels without knowledge of the sequences involved, thereby decreasing a major portion of the overall sample preparation cost. The reaction employs two thermostable enzymes, BST-polymerase and duplex-specific nuclease DSN. DSN initiates random 'nicks' on double-stranded-DNA which enable BST to polymerize DNA by displacing the nicked-strand. Displaced strands re-hybridize and the process leads to an exponential chain-reaction generating biotinylated DNA fragments within minutes. When starting from single-stranded-DNA, DNA is first converted to double-stranded-DNA via terminal-deoxynucleotidyl-transferase (TdT) prior to initiation of BST-DSN reaction. Biotinylated probes generated by TdT-BST-DSN (TBD) reactions using panels of 33, 190 or 7186 DNA targets are used for hybrid-capture-based target enrichment from amplified circulating-DNA, followed by targeted re-sequencing. Polymerase-nuclease isothermal-chain-reactions generate random amplified probes with no apparent sequence dependence. One round of target-capture using TBD probes generates a modest on-target sequencing ratio, while two successive rounds of capture generate >80% on-target reads with good sequencing uniformity. TBD-reactions generate enough capture-probes to increase by approximately two to three orders-of-magnitude the target-enrichment experiments possible from an initial set of probes.

Multiplexed Elimination of Wild-Type DNA and High-Resolution Melting Prior to Targeted Resequencing of Liquid Biopsies.

BACKGROUND: The use of clinical samples and circulating cell-free DNA (cfDNA) collected from liquid biopsies for diagnostic and prognostic applications in cancer is burgeoning, and improved methods that reduce the influence of excess wild-type (WT) portion of the sample are desirable. Here we present enrichment of mutation-containing sequences using enzymatic degradation of WT DNA. Mutation enrichment is combined with high-resolution melting (HRM) performed in multiplexed closed-tube reactions as a rapid, cost-effective screening tool before targeted resequencing. METHODS: We developed a homogeneous, closed-tube approach to use a double-stranded DNA-specific nuclease for degradation of WT DNA at multiple targets simultaneously. The No Denaturation Nuclease-assisted Minor Allele Enrichment with Probe Overlap (ND-NaME-PrO) uses WT oligonucleotides overlapping both strands on putative DNA targets. Under conditions of partial denaturation (DNA breathing), the oligonucleotide probes enhance double-stranded DNA-specific nuclease digestion at the selected targets, with high preference toward WT over mutant DNA. To validate ND-NaME-PrO, we used multiplexed HRM, digital PCR, and MiSeq targeted resequencing of mutated genomic DNA and cfDNA. RESULTS: Serial dilution of KRAS mutation-containing DNA shows mutation enrichment by 10- to 120-fold and detection of allelic fractions down to 0.01%. Multiplexed ND-NaME-PrO combined with multiplexed PCR-HRM showed mutation scanning of 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 were enriched up to approximately 100-fold (average approximately 25-fold) and identified via targeted resequencing. CONCLUSIONS: 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 prescreening before targeted resequencing.

A Size-Selective Intracellular Delivery Platform.

Identifying and separating a subpopulation of cells from a heterogeneous mixture are essential elements of biological research. Current approaches require detailed knowledge of unique cell surface properties of the target cell population. A method is described that exploits size differences of cells to facilitate selective intracellular delivery using a high throughput microfluidic device. Cells traversing a constriction within this device undergo a transient disruption of the cell membrane that allows for cytoplasmic delivery of cargo. Unique constriction widths allow for optimization of delivery to cells of different sizes. For example, a 4 µm wide constriction is effective for delivery of cargo to primary human T-cells that have an average diameter of 6.7 µm. In contrast, a 6 or 7 µm wide constriction is best for large pancreatic cancer cell lines BxPc3 (10.8 µm) and PANC-1 (12.3 µm). These small differences in cell diameter are sufficient to allow for selective delivery of cargo to pancreatic cancer cells within a heterogeneous mixture containing T-cells. The application of this approach is demonstrated by selectively delivering dextran-conjugated fluorophores to circulating tumor cells in patient blood allowing for their subsequent isolation and genomic characterization.

Detection of Copy-Number Variation in Circulating Cell-Free DNA in Patients With Uveal Melanoma.

PURPOSE: Somatic chromosomal alterations, particularly monosomy 3 and 8q gains, have been associated with metastatic risk in uveal melanoma (UM). Whole genome-scale evaluation of detectable alterations in cell-free DNA (cfDNA) in UM could provide valuable prognostic information. Our pilot study evaluates the correlation between genomic information using ultra-low-pass whole-genome sequencing (ULP-WGS) of cfDNA in UM and associated clinical outcomes. MATERIALS AND METHODS: ULP-WGS of cfDNA was performed on 29 plasma samples from 16 patients, 14 metastatic UM (mUM) and two non-metastatic, including pre- and post-treatment mUM samples from 10 patients treated with immunotherapy and one with liver-directed therapy. We estimated tumor fraction (TFx) and detected copy-number alterations (CNAs) using ichorCNA. Presence of 8q amplification was further analyzed using the likelihood ratio test (LRT). RESULTS: Eleven patients with mUM (17 samples) of 14 had detectable circulating tumor DNA (ctDNA). 8q gain was detected in all 17, whereas monosomy 3 was detectable in 10 of 17 samples. TFx generally correlated with disease status, showing an increase at the time of disease progression (PD). 8q gain detection sensitivity appeared greater with the LRT than with ichorCNA at lower TFxs. The only patient with mUM with partial response on treatment had a high pretreatment TFx and undetectable on-treatment ctDNA, correlating with her profound response and durable survival. CONCLUSION: ctDNA can be detected in mUM using ULP-WGS, and the TFx correlates with DS. 8q gain was consistently detectable in mUM, in line with previous studies indicating 8q gains early in primary UM and higher amplification with PD. Our work suggests that detection of CNAs by ULP-WGS, particularly focusing on 8q gain, could be a valuable blood biomarker to monitor PD in UM.

FinaleMe: Predicting DNA methylation by the fragmentation patterns of plasma cell-free DNA.

Analysis of DNA methylation in cell-free DNA (cfDNA) reveals clinically relevant biomarkers but requires specialized protocols and sufficient input material that limits its applicability. Millions of cfDNA samples have been profiled by genomic sequencing. To maximize the gene regulation information from the existing dataset, we developed FinaleMe, a non-homogeneous Hidden Markov Model (HMM), to predict DNA methylation of cfDNA and, therefore, tissues-of-origin directly from plasma whole-genome sequencing (WGS). We validated the performance with 80 pairs of deep and shallow-coverage WGS and whole-genome bisulfite sequencing (WGBS) data.

Assay Validation of Cell-Free DNA Shallow Whole-Genome Sequencing to Determine Tumor Fraction in Advanced Cancers.

Blood-based liquid biopsy is increasingly used in clinical care of patients with cancer, and fraction of tumor-derived DNA in circulation (tumor fraction; TFx) has demonstrated clinical validity across multiple cancer types. To determine TFx, shallow whole-genome sequencing of cell-free DNA (cfDNA) can be performed from a single blood sample, using an established computational pipeline (ichorCNA), without prior knowledge of tumor mutations, in a highly cost-effective manner. We describe assay validation of this approach to facilitate broad clinical application, including evaluation of assay sensitivity, precision, repeatability, reproducibility, pre-analytic factors, and DNA quality/quantity. Sensitivity to detect TFx of 3% (lower limit of detection) was 97.2% to 100% at 1× and 0.1× mean sequencing depth, respectively. Precision was demonstrated on distinct sequencing instruments (HiSeqX and NovaSeq) with no observable differences. The assay achieved prespecified 95% agreement of TFx across replicates of the same specimen (repeatability) and duplicate samples in different batches (reproducibility). Comparison of samples collected in EDTA and Streck tubes from single venipuncture in 23 patients demonstrated that EDTA or Streck tubes were comparable if processed within 8 hours. On the basis of a range of DNA inputs (1 to 50 ng), 20 ng cfDNA is the preferred input, with 5 ng minimum acceptable. Overall, this shallow whole-genome sequencing of cfDNA and ichorCNA approach offers sensitive, precise, and reproducible quantitation of TFx, facilitating assay application in clinical cancer care.

FinaleMe: Predicting DNA methylation by the fragmentation patterns of plasma cell-free DNA.

Analysis of DNA methylation in cell-free DNA reveals clinically relevant biomarkers but requires specialized protocols such as whole-genome bisulfite sequencing. Meanwhile, millions of cell-free DNA samples are being profiled by whole-genome sequencing. Here, we develop FinaleMe, a non-homogeneous Hidden Markov Model, to predict DNA methylation of cell-free DNA and, therefore, tissues-of-origin, directly from plasma whole-genome sequencing. We validate the performance with 80 pairs of deep and shallow-coverage whole-genome sequencing and whole-genome bisulfite sequencing data.

Priming agents transiently reduce the clearance of cell-free DNA to improve liquid biopsies.

Liquid biopsies enable early detection and monitoring of diseases such as cancer, but their sensitivity remains limited by the scarcity of analytes such as cell-free DNA (cfDNA) in blood. Improvements to sensitivity have primarily relied on enhancing sequencing technology ex vivo. We sought to transiently augment the level of circulating tumor DNA (ctDNA) in a blood draw by attenuating its clearance in vivo. We report two intravenous priming agents given 1 to 2 hours before a blood draw to recover more ctDNA. Our priming agents consist of nanoparticles that act on the cells responsible for cfDNA clearance and DNA-binding antibodies that protect cfDNA. In tumor-bearing mice, they greatly increase the recovery of ctDNA and improve the sensitivity for detecting small tumors.

A nanoparticle priming agent reduces cellular uptake of cell-free DNA and enhances the sensitivity of liquid biopsies.

Liquid biopsies are enabling minimally invasive monitoring and molecular profiling of diseases across medicine, but their sensitivity remains limited by the scarcity of cell-free DNA (cfDNA) in blood. Here, we report an intravenous priming agent that is given prior to a blood draw to increase the abundance of cfDNA in circulation. Our priming agent consists of nanoparticles that act on the cells responsible for cfDNA clearance to slow down cfDNA uptake. In tumor-bearing mice, this agent increases the recovery of circulating tumor DNA (ctDNA) by up to 60-fold and improves the sensitivity of a ctDNA diagnostic assay from 0% to 75% at low tumor burden. We envision that this priming approach will significantly improve the performance of liquid biopsies across a wide range of clinical applications in oncology and beyond.

Single duplex DNA sequencing with CODEC detects mutations with high sensitivity.

Detecting mutations from single DNA molecules is crucial in many fields but challenging. Next-generation sequencing (NGS) affords tremendous throughput but cannot directly sequence double-stranded DNA molecules ('single duplexes') to discern the true mutations on both strands. Here we present Concatenating Original Duplex for Error Correction (CODEC), which confers single duplex resolution to NGS. CODEC affords 1,000-fold higher accuracy than NGS, using up to 100-fold fewer reads than duplex sequencing. CODEC revealed mutation frequencies of 2.72 × 10-8 in sperm of a 39-year-old individual, and somatic mutations acquired with age in blood cells. CODEC detected genome-wide, clonal hematopoiesis mutations from single DNA molecules, single mutated duplexes from tumor genomes and liquid biopsies, microsatellite instability with 10-fold greater sensitivity and mutational signatures, and specific tumor mutations with up to 100-fold fewer reads. CODEC enables more precise genetic testing and reveals biologically significant mutations, which are commonly obscured by NGS errors.

Circulating tumor DNA association with residual cancer burden after neoadjuvant chemotherapy in triple-negative breast cancer in TBCRC 030.

Purpose: To examine circulating tumor DNA (ctDNA) and its association with residual cancer burden (RCB) using an ultrasensitive assay in patients with triple-negative breast cancer (TNBC) receiving neoadjuvant chemotherapy (NAT). Patients and Methods: We identified responders (RCB-0/1) and matched non-responders (RCB-2/3) from the phase II TBCRC 030 prospective study of neoadjuvant paclitaxel vs. cisplatin in TNBC. We collected plasma samples at baseline, three weeks, and twelve weeks (end of therapy). We created personalized ctDNA assays utilizing MAESTRO mutation enrichment sequencing. We explored associations between ctDNA and RCB status and disease recurrence. Results: Of 139 patients, 68 had complete samples and no additional NAT. Twenty-two were responders and 19 of those had sufficient tissue for whole-genome sequencing. We identified an additional 19 non-responders for a matched case-control analysis of 38 patients using a MAESTRO ctDNA assay tracking 319-1000 variants (median 1000) to 114 plasma samples from 3 timepoints. Overall, ctDNA positivity was 100% at baseline, 79% at week 3, and 55% at week 12. Median tumor fraction (TFx) was 3.7 × 10 -4 (range: 7.9 × 10 -7 to 4.9 × 10 -1 ). TFx decreased 285-fold from baseline to week 3 in responders and 24-fold in non-responders. Week 12 ctDNA clearance correlated with RCB: clearance was observed in 10/11 patients with RCB-0, 3/8 with RCB-1, 4/15 with RCB-2, and 0/4 with RCB-3. Among 6 patients with known recurrence five had persistent ctDNA at week 12. Conclusion: NAT for TNBC reduced ctDNA TFx by 285-fold in responders and 24-fold in non-responders. In 58% (22/38) of patients, ctDNA TFx dropped below the detection level of a commercially available test, emphasizing the need for sensitive tests. Additional studies will determine if ctDNA-guided approaches can improve outcomes.

Liquid biopsy detection of genomic alterations in pediatric brain tumors from cell-free DNA in peripheral blood, CSF, and urine.

BACKGROUND: The ability to identify genetic alterations in cancers is essential for precision medicine; however, surgical approaches to obtain brain tumor tissue are invasive. Profiling circulating tumor DNA (ctDNA) in liquid biopsies has emerged as a promising approach to avoid invasive procedures. Here, we systematically evaluated the feasibility of profiling pediatric brain tumors using ctDNA obtained from plasma, cerebrospinal fluid (CSF), and urine. METHODS: We prospectively collected 564 specimens (257 blood, 240 urine, and 67 CSF samples) from 258 patients across all histopathologies. We performed ultra-low-pass whole-genome sequencing (ULP-WGS) to assess copy number variations and estimate tumor fraction and developed a pediatric CNS tumor hybrid capture panel for deep sequencing of specific mutations and fusions. RESULTS: ULP-WGS detected copy number alterations in 9/46 (20%) CSF, 3/230 (1.3%) plasma, and 0/153 urine samples. Sequencing detected alterations in 3/10 (30%) CSF, 2/74 (2.7%) plasma, and 0/2 urine samples. The only positive results were in high-grade tumors. However, most samples had insufficient somatic mutations (median 1, range 0-39) discoverable by the sequencing panel to provide sufficient power to detect tumor fractions of greater than 0.1%. CONCLUSIONS: Children with brain tumors harbor very low levels of ctDNA in blood, CSF, and urine, with CSF having the most DNA detectable. Molecular profiling is feasible in a small subset of high-grade tumors. The level of clonal aberrations per genome is low in most of the tumors, posing a challenge for detection using whole-genome or even targeted sequencing methods. Substantial challenges therefore remain to genetically characterize pediatric brain tumors from liquid biopsies.

Massively parallel enrichment of low-frequency alleles enables duplex sequencing at low depth.

Assaying for large numbers of low-frequency mutations requires sequencing at extremely high depth and accuracy. Increasing sequencing depth aids the detection of low-frequency mutations yet limits the number of loci that can be simultaneously probed. Here we report a method for the accurate tracking of thousands of distinct mutations that requires substantially fewer reads per locus than conventional hybrid-capture duplex sequencing. The method, which we named MAESTRO (for minor-allele-enriched sequencing through recognition oligonucleotides), combines massively parallel mutation enrichment with duplex sequencing to track up to 10,000 low-frequency mutations, with up to 100-fold fewer reads per locus. We show that MAESTRO can be used to test for chimaerism by tracking donor-exclusive single-nucleotide polymorphisms in sheared genomic DNA from human cell lines, to validate whole-exome sequencing and whole-genome sequencing for the detection of mutations in breast-tumour samples from 16 patients, and to monitor the patients for minimal residual disease via the analysis of cell-free DNA from liquid biopsies. MAESTRO improves the breadth, depth, accuracy and efficiency of mutation testing by sequencing.

A framework for clinical cancer subtyping from nucleosome profiling of cell-free DNA.

Cell-free DNA (cfDNA) has the potential to inform tumor subtype classification and help guide clinical precision oncology. Here we develop Griffin, a framework for profiling nucleosome protection and accessibility from cfDNA to study the phenotype of tumors using as low as 0.1x coverage whole genome sequencing data. Griffin employs a GC correction procedure tailored to variable cfDNA fragment sizes, which generates a better representation of chromatin accessibility and improves the accuracy of cancer detection and tumor subtype classification. We demonstrate estrogen receptor subtyping from cfDNA in metastatic breast cancer. We predict estrogen receptor subtype in 139 patients with at least 5% detectable circulating tumor DNA with an area under the receive operator characteristic curve (AUC) of 0.89 and validate performance in independent cohorts (AUC = 0.96). In summary, Griffin is a framework for accurate tumor subtyping and can be generalizable to other cancer types for precision oncology applications.