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.

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).

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.

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.

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.

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.

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%.

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.

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.

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.