Biology subjects

Adalsteinsson, V. A.

Publications and source records attributed to Adalsteinsson, V. A..

9 recordsLinked to original sources

bioRxiv2026

Molecular determinants of antibody-mediated priming to enhance detection of ctDNA

Liquid biopsies can enable cancer detection and monitoring yet remain limited by low concentrations of ctDNA. To address this limitation, we previously introduced a monoclonal antibody (mAb) "priming agent" that transiently increased the concentration of ctDNA in blood. Here, we investigated the molecular features that drive this effect. In a panel of novel mAbs that bound cfDNA, both those targeting dsDNA and those targeting mononucleosomes increased the concentration of ctDNA. mAbs with high avidity to dsDNA performed best, suggesting dsDNA as the key binding target. One agent preserved short, biologically informative molecules of cfDNA typically depleted at baseline. The Fc domain was dispensable, as F(ab)2 fragments retained priming activity. Leveraging these insights, we engineered single-chain agents using the dsDNA binding domain sso7d, expanding priming strategies beyond immunoglobulins. This study identifies the molecular features and general design principles for mAb-based priming agents to enhance recovery and detection of ctDNA. SignificanceLow concentration of ctDNA limits the sensitivity of liquid biopsies in many applications. This study shows how engineered antibodies and other dsDNA-binding molecules can inhibit clearance of ctDNA from the bloodstream and increase concentration of ctDNA in a blood draw, and identifies the key features that enable this activity. Modulation of cfDNA in the bloodstream using these agents can increase recovery of ctDNA and improve the performance of liquid biopsies for cancer detection.

bioengineering↗

bioRxiv2025

MEDUSA: Maintaining Entire DNA Duplexes for Utmost Sequencing Accuracy

Highly accurate DNA sequencing is essential for many applications. Duplex sequencing-based methods achieve unparalleled accuracy by requiring reads from both strands of the original DNA duplex to match. Yet, methods to prepare dsDNA for sequencing may resynthesize portions of each DNA duplex and cause base damage errors on one strand to become indistinguishable from true mutations on both strands. Here, we report MEDUSA (Maintaining Entire DNA Duplexes for Utmost Sequencing Accuracy) which minimizes dsDNA resynthesis to maximize duplex sequencing accuracy and yield. MEDUSA carefully repairs and blunts fragmented dsDNA, then employs apyrase to digest residual dNTPs, followed by restricted dA-tailing to prevent resynthesis. MEDUSA affords full genome coverage in a simplified protocol that is broadly compatible with dsDNA fragmentation and library preparation kits. We benchmarked MEDUSA on sheared genomic DNA and cell-free DNA and found a residual SNV frequency within a median 1.23-fold (range 0.92-1.88; p < 0.001) of what was expected if resynthesis was mostly blocked, but with full genome coverage and duplex yields within a median 1.02-fold (range 0.33 - 1.46; p = 0.258) of traditional methods that do not limit resynthesis. In all, MEDUSA could enable high breadth or depth of duplex sequencing while limiting false discovery. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/687202v1_ufig1.gif" ALT="Figure 1"> View larger version (12K): org.highwire.dtl.DTLVardef@1ddbc77org.highwire.dtl.DTLVardef@802ca9org.highwire.dtl.DTLVardef@f443ecorg.highwire.dtl.DTLVardef@974bdb_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2025

Genome-wide extraction of differentially methylated DNA regions using adapter-anchored proximity primers

The epigenetic deregulation of CpG islands (CGIs) plays a crucial role in cancer initiation and progression. CGIs comprise 1-2% of the human genome and are rich in differentially methylated regions (DMRs) that can serve as cancer biomarkers in clinical samples and liquid biopsies. Focusing epigenetic sequencing on CpG-rich sequences, including CGIs and avoiding non-informative regions, offers an efficient and sensitive approach for cancer identification and tracking, especially within samples containing excess of unaltered, normal DNA. To this end, we have developed Adaptor-anchored Methylation amplification via Proximity Primers (aMAPP), a versatile PCR-based enrichment method. aMAPP employs specially designed primers to selectively enrich either methylated or unmethylated CpGs, depending on the upstream methylation conversion method employed. aMAPP achieves high coverage of genome-wide CGIs and detects hundreds of DMRs in tumor samples compared to adjacent normal tissue using ultra-low depth sequencing ([~]300,000 reads). It enables tracing of aberrant methylation down to allelic frequency 0.01% in dilutions of tumor DNA and in cell-free DNA samples, can be applied using picogram amounts of DNA, and can be adapted to enrich either small panels of cancer-specific DMRs, or the majority (>90%) of genomic CGIs and CpGs. aMAPP offers a simple, cost-effective, and highly sensitive approach for capturing the epigenetic footprint of genome-wide CpGs and identifying aberrantly methylated or un-methylated genomic regions. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/660377v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@b8b303org.highwire.dtl.DTLVardef@1f0a70corg.highwire.dtl.DTLVardef@67391dorg.highwire.dtl.DTLVardef@d6ac75_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

Methyl-CODEC enables simultaneous methylation and duplex sequencing

DNA mutations and methylation often contribute to disease development in a synergistic manner. While duplex sequencing is the most accurate method for detecting DNA mutations, it typically lacks the ability to simultaneously assess methylation or requires many reads. Here, we developed Methyl-CODEC to enable simultaneous methylation sequencing and duplex sequencing using single read pairs. To achieve this, Methyl-CODEC links an enzymatically deaminated sense strand to the reverse complement of the antisense strand, which is protected from conversion by using conversion-resistant dCTPs in the strand linking step of CODEC. Methyl-CODEC shows high concordance with standard enzymatic or bisulfite based whole genome methylation sequencing, while also uniquely preserving the original DNA sequence. This improves genetic sequencing accuracy, enables better alignment for next-generation sequencing (NGS), and distinguishes C>T mutations from unmethylated Cs. It also identifies rare mutations including those producing methylated Cs, which are enriched in CpG contexts. Methyl-CODEC opens new horizons for enhanced detection of biomarkers in cancer and molecular medicine.

genomics↗

bioRxiv2024

DirectHRD enables sensitive scar-based classification of homologous recombination deficiency (HRD)

Homologous recombination deficiency (HRD) is a predictive biomarker for efficacy of PARP inhibition and platinum chemotherapy but remains challenging to detect from low tumor fraction samples such as liquid biopsies. Here, we describe DirectHRD, a whole-genome sequencing (WGS) scar-based classifier that is 10x more sensitive than state-of-the-art methods. DirectHRD can detect HRD at >=1% tumor fraction using 50x WGS of cell-free DNA.

bioinformatics↗

bioRxiv2024

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.

bioinformatics↗

bioRxiv2021

CODEC enables 'single duplex' sequencing

Detecting mutations as rare as a single molecule is crucial in many fields such as cancer diagnostics and aging research but remains challenging. Third generation sequencers can read a double-stranded DNA molecule (a single duplex) in whole to identify true mutations on both strands apart from false mutations on either strand but with limited accuracy and throughput. Although next generation sequencing (NGS) can track dissociated strands with Duplex Sequencing, the need to sequence each strand independently severely diminishes its throughput. Here, we developed a hybrid method called Concatenating Original Duplex for Error Correction (CODEC) that combines the massively parallel nature of NGS with the single-molecule capability of third generation sequencing. CODEC physically links both strands to enable NGS to sequence a single duplex with a single read pair. By comparing CODEC and Duplex Sequencing, we showed that CODEC achieved a similar error rate (10-6) with 100 times fewer reads and conferred single duplex resolution to most major NGS workflows.

bioengineering↗

bioRxiv2021

MAESTRO affords 'breadth and depth' for mutation testing

The ability to assay large numbers of low-abundance mutations is crucial in biomedicine. Yet, the technical hurdles of sequencing multiple mutations at extremely high depth and accuracy remain daunting. For sequencing low-level mutations, its either depth or breadth but not both. Here, we report a simple and powerful approach to accurately track thousands of distinct mutations with minimal reads. Our technique called MAESTRO (minor allele enriched sequencing through recognition oligonucleotides) employs massively-parallel mutation enrichment to empower duplex sequencing--one of the most accurate methods--to track up to 10,000 low-frequency mutations with up to 100-fold less sequencing. In example use cases, we show that MAESTRO could enable mutation validation from cancer genome sequencing studies. We also show that it could track thousands of mutations from a patients tumor in cell-free DNA, which may improve detection of minimal residual disease from liquid biopsies. In all, MAESTRO improves the breadth, depth, accuracy, and efficiency of mutation testing.

molecular biology↗

bioRxiv2019

Reversion and non-reversion mechanisms of resistance to PARP inhibitor or platinum chemotherapy in BRCA1/2-mutant metastatic breast cancer

BackgroundLittle is known about mechanisms of resistance to PARP inhibitors and platinum chemotherapy in patients with metastatic breast cancer and BRCA1/2 mutations. Further investigation of resistance in clinical cohorts may point to strategies to prevent or overcome treatment failure. Patients and MethodsWe obtained tumor biopsies from metastatic breast cancer patients with BRCA1/2 deficiency before and after acquired resistance to PARP inhibitor or platinum chemotherapy. Whole exome sequencing was performed on each tumor, germline DNA, and circulating tumor DNA. Tumors underwent RNA sequencing, and immunohistochemical staining for RAD51 foci on tumor sections was performed for functional assessment of intact homologous recombination. ResultsPre- and post-resistance tumor samples were sequenced from 8 patients (4 with BRCA1 and 4 with BRCA2 mutation; 4 treated with PARP inhibitor and 4 with platinum). Following disease progression on DNA-damaging therapy, four patients (50%) acquired at least one somatic reversion alteration likely to result in functional BRCA1/2 protein detected by tumor or circulating tumor DNA sequencing. Two patients with germline BRCA1 deficiency acquired genomic alterations anticipated to restore homologous recombination through increased DNA end resection: loss of TP53BP1 in one patient and amplification of MRE11A in another. RAD51 foci were acquired post-resistance in all patients with genomic reversion, consistent with reconstitution of homologous recombination. All patients whose tumors demonstrated RAD51 foci post-resistance were intrinsically resistant to subsequent lines of DNA-damaging therapy. ConclusionsGenomic reversion in BRCA1/2 was the most commonly observed mechanism of resistance, occurring in 4 of 8 patients. Novel sequence alterations leading to increased DNA end resection were seen in two patients, and may be targetable for therapeutic benefit. The presence of RAD51 foci by immunohistochemistry was consistent with BRCA1/2 protein functional status from genomic data and predicted response to later DNA-damaging therapy, supporting RAD51 focus formation as a clinically useful biomarker.

cancer biology↗