DNA Nanoswitch Barcodes for Multiplexed Biomarker Profiling.
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ABSTRACT:
SUBMITTER: Chandrasekaran AR
PROVIDER: S-EPMC8059342 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundHigh-throughput sequencing technologies are improving in quality, capacity and costs, providing versatile applications in DNA and RNA research. For small genomes or fraction of larger genomes, DNA samples can be mixed and loaded together on the same sequencing track. This so-called multiplexing approach relies on a specific DNA tag or barcode that is attached to the sequencing or amplification primer and hence appears at the beginning of the sequence in every read. After sequencing, each sample read is identified on the basis of the respective barcode sequence.Alterations of DNA barcodes during synthesis, primer ligation, DNA amplification, or sequencing may lead to incorrect sample identification unless the error is revealed and corrected. This can be accomplished by implementing error correcting algorithms and codes. This barcoding strategy increases the total number of correctly identified samples, thus improving overall sequencing efficiency. Two popular sets of error-correcting codes are Hamming codes and Levenshtein codes.ResultLevenshtein codes operate only on words of known length. Since a DNA sequence with an embedded barcode is essentially one continuous long word, application of the classical Levenshtein algorithm is problematic. In this paper we demonstrate the decreased error correction capability of Levenshtein codes in a DNA context and suggest an adaptation of Levenshtein codes that is proven of efficiently correcting nucleotide errors in DNA sequences. In our adaption we take the DNA context into account and redefine the word length whenever an insertion or deletion is revealed. In simulations we show the superior error correction capability of the new method compared to traditional Levenshtein and Hamming based codes in the presence of multiple errors.ConclusionWe present an adaptation of Levenshtein codes to DNA contexts capable of correction of a pre-defined number of insertion, deletion, and substitution mutations. Our improved method is additionally capable of recovering the new length of the corrupted codeword and of correcting on average more random mutations than traditional Levenshtein or Hamming codes.As part of this work we prepared software for the flexible generation of DNA codes based on our new approach. To adapt codes to specific experimental conditions, the user can customize sequence filtering, the number of correctable mutations and barcode length for highest performance.
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Project description:Our understanding of how genotype controls phenotype is limited by the scale at which we can precisely alter the genome and assess the phenotypic consequences of each perturbation. Here we describe a CRISPR-Cas9-based method for multiplexed accurate genome editing with short, trackable, integrated cellular barcodes (MAGESTIC) in Saccharomyces cerevisiae. MAGESTIC uses array-synthesized guide-donor oligos for plasmid-based high-throughput editing and features genomic barcode integration to prevent plasmid barcode loss and to enable robust phenotyping. We demonstrate that editing efficiency can be increased more than fivefold by recruiting donor DNA to the site of breaks using the LexA-Fkh1p fusion protein. We performed saturation editing of the essential gene SEC14 and identified amino acids critical for chemical inhibition of lipid signaling. We also constructed thousands of natural genetic variants, characterized guide mismatch tolerance at the genome scale, and ascertained that cryptic Pol III termination elements substantially reduce guide efficacy. MAGESTIC will be broadly useful to uncover the genetic basis of phenotypes in yeast.
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Project description:Epigenetics, such as the dynamic interplay between DNA methylation and demethylation, play diverse roles in critical cellular events. Enzymatic activity at CpG sites, where cytosines are methylated or demethylated, is known to be influenced by the density of CpGs, methylation states, and the flanking sequences of a CpG site. However, how the relevant enzymes are recruited to and recognize their target DNA is less clear. Moreover, although DNA-binding epigenetic enzymes are ideal targets for therapeutic intervention, these targets have been rarely exploited. Single-molecule techniques offer excellent capabilities to probe site-specific protein-DNA interactions and unravel the dynamics. Here, we develop a single-molecule approach that allows multiplexed profiling of protein-DNA complexes using magnetic tweezers. When a DNA hairpin with multiple binding sites is unzipping, strand separation pauses at the positions bound by a protein. We can thus measure site-specific binding probabilities and dissociation time directly. Taking the TET1 CXXC domain as an example, we show that TET1 CXXC binds multiple CpG motifs with various flanking nucleotides or different methylation patterns in an AT-rich DNA. We are able to establish for the first time, at nanometer resolution, that TET1 CXXC prefers G/C flanked CpG motif over C/G, A/T, or T/A flanked ones. CpG methylation strengthens TET1 CXXC recruitment but has little effect on dissociation time. Finally, we demonstrate that TET1 CXXC can distinguish five CpG clusters in a CpG island with crowded binding motifs. We anticipate that the feasibility of single-molecule multiplexed profiling assays will contribute to the understanding of protein-DNA interactions.
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Project description:Suspension arrays based on optical encoded microspheres have attracted great attention for multiplexed detection in gene analysis, protein profiling, early disease diagnosis, treatment monitoring and so on. However, the fluorescence stability of barcodes and detection sensitivity require further improvement to meet the increasing demands of "precision diagnosis". Methods: This work reports a novel suspension array platform based on extremely stable AIEgens (AIE33 and AIE NIR800) microbeads as barcodes and AIEgens (1,1,2,3,4,5-Hexaphenyl-1H-silole, HPS) nanobeads as fluorescent signal reporter coupled with flow cytometry for multiplexed detection. Results: Due to the excellent fluorescent signal amplification effect of the HPS nanobeads, our multiplex assay showed enhanced detection sensitivity, compared to multiplex assay using QDs nanobeads (up to 3-fold improvement) and commercial organic dye of phycoerythrin (up to 5-fold improvement) as the fluorescent signal reporters. Conclusion: Furthermore, validating experiments showed similar detection performance to the clinical gold-standard method of ImmunoCAP for allergen detection in patient serum samples, demonstrating the suspension array platform based on AIEgens microbeads with excellent fluorescence stability and AIEgens nanobeads with strong signal amplification ability is promising for high-sensitivity multiplexed bioassay applications.
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Project description:BackgroundThe fibroblast growth factor receptor 4 (FGFR4) pathway is an essential regulatory component of bile acid synthesis, and its relationship with hepatocellular carcinoma (HCC) has been reported. We investigated the gene expression and clinical significance of FGFR4 and related pathways in intrahepatic cholangiocarcinoma (iCCA).ResultsThe median age was 56 years (range 30-78) and 34 patients (74%) were male. Six patients (13%) had hepatitis B virus infection, with or without liver cirrhosis. Overall survival was significantly associated with FGFR4 (p = 0.004), FGF19 (p = 0.047), FGF21 (p = 0.04), and KLB (p = 0.03) expression. In the multivariate analysis with potential prognostic factors, high expression of FGF19, FGF21, and FGFR4 was significantly associated with better survival. In the analysis using the TCGA iCCA dataset, mRNA overexpression of at least 1 of the FGFR4-related genes was significantly associated with better disease-free survival (p = 0.02).Materials and methodsWe assessed the expression of 98 genes in formalin-fixed paraffin embedded tumor tissue specimens from 46 patients with surgically resected iCCA using a NanoString platform. This included 10 FGF pathway genes (e.g. FGFR1-4, KLB, FGF3, 4, 19, 21, and 23), 19 distal marker genes (e.g. CYP7A1 and CYP17A1), 31 genes relevant to HCC and iCCA (e.g. AFP, TS), 18 copy number variation matched genes, and 20 control genes. Log-transformation of gene expression was performed for normalization and statistical analysis. Overall survival was correlated with gene expression (< median vs. ≥ median) using a log-rank test. The prognostic impact of FGFR4-related genes was validated using the public TCGA dataset for iCCA.ConclusionsOur results indicate that mRNA expression of FGFR4-related genes may be a biomarker to define the distinctive molecular phenotype of iCCA. Future preclinical and clinical validation is required to define the role of the FGFR4 pathway in iCCA.
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