Multiple-primer DNA sequencing method.

A multiple-primer DNA sequencing approach suitable for genotyping, detection and identification of microorganisms and viruses has been developed. In this new method two or more sequencing primers, combined in a pool, are added to a DNA sample of interest. The oligonucleotide that hybridizes to the DNA sample will function as a primer during the subsequent DNA sequencing procedure. This strategy is suited for selective detection and genotyping of relevant microorganisms and samples harboring different DNA targets such as multiple variant/infected samples as well as unspecific amplification products. This method is used here in a model system for detection and typing of high-risk oncogenic human papilloma viruses (HPVs) in samples containing multiple infections/variants or unspecific amplification products. Type-specific sequencing primers were designed for four of the most oncogenic (high-risk) HPV types (HPV-16, HPV-18, HPV-33, and HPV-45). The primers were combined and added to a sample containing a mixture of one high-risk (16, 18, 33, or 45) and one or two low-risk types. The DNA samples were sequenced by the Pyrosequencing technology and the Sanger dideoxy sequencing method. Correct genotyping was achieved in all tested combinations. This multiple-sequencing primer approach also improved the sequence data quality for samples containing unspecific amplification products. The new strategy is highly suitable for diagnostic typing of relevant species/genotypes of microorganisms.     

Pyrosequencing‐based strategy for a successful SNP detection in two hypervariable regions: HV‐I/HV‐II of the human mitochondrial displacement loop

Forensic analysis of mitochondrial displacement loop (D‐loop) sequences using Sanger sequencing or SNP detection by minisequencing is well established. Pyrosequencing has become an important alternative because it enables high‐throughput analysis and the quantification of individual mitochondrial DNAs (mtDNAs) in samples originating from more than one individual. DNA typing of the mitochondrial D‐loop region is usually the method of choice if STR analysis fails because of trace amounts of DNA and/or extensive degradation. The main aim of the present work was to optimize the efficiency of pyrosequencing. To do this, 31 SNPs within the hypervariable regions I and II of the D‐loop of human mtDNA were simultaneously analyzed. As a novel approach, we applied two sets of amplification primers for the multiplexing assay. These went in combination with four sequencing primers for pyrosequencing. This method was compared with conventional sequencing of mtDNA from blood and biological trace materials.     

Nanopore sequencing: An enrichment‐free alternative to mitochondrial DNA sequencing

Mitochondrial DNA sequence data are often utilized in disease studies, conservation genetics and forensic identification. The current approaches for sequencing the full mtGenome typically require several rounds of PCR enrichment during Sanger or MPS protocols followed by fairly tedious assembly and analysis. Here we describe an efficient approach to sequencing directly from genomic DNA samples without prior enrichment or extensive library preparation steps. A comparison is made between libraries sequenced directly from native DNA and the same samples sequenced from libraries generated with nine overlapping mtDNA amplicons on the Oxford Nanopore MinION? device. The native and amplicon library preparation methods and alternative base calling strategies were assessed to establish error rates and identify trends of discordance between the two library preparation approaches. For the complete mtGenome, 16 569 nucleotides, an overall error rate of approximately 1.00% was observed. As expected with mtDNA, the majority of error was detected in homopolymeric regions. The use of a modified basecaller that corrects for ambiguous signal in homopolymeric stretches reduced the error rate for both library preparation methods to approximately 0.30%. Our study indicates that direct mtDNA sequencing from native DNA on the MinION? device provides comparable results to those obtained from common mtDNA sequencing methods and is a reliable alternative to approaches using PCR‐enriched libraries.     

Targeted next-generation sequencing by specific capture of multiple genomic loci using low-volume microfluidic DNA arrays

We report a flexible method for selective capture of sequence fragments from complex, eukaryotic genome libraries for next-generation sequencing based on hybridization to DNA microarrays. Using microfluidic array architecture and integrated hardware, the process is amenable to complete automation and does not introduce amplification steps into the standard library preparation workflow, thereby avoiding bias of sequence distribution and fragment lengths. We captured a discontiguous human genomic target region of 185 kb using a tiling design with 50mer probes. Analysis by high-throughput sequencing using an Illumina/Solexa 1G Genome Analyzer revealed 2150-fold enrichment with mean per base coverage between 4.6 and 107.5-fold for the individual target regions. This method represents a flexible and cost-effective approach for large-scale resequencing of complex genomes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Stephan Bau and Nadine Schracke contributed equally to this work.     

Differences between predicted and observed sequences in Saccharomyces cerevisiae

We recently studied the protein composition of a Saccharomyces cerevisiae wine yeast strain (K310) of enological interest. About 2,500 spots of 8-250 kDa observed molecular mass were resolved by two-dimensional gel electrophoresis. Experimental molecular masses and isoelectric points were calculated for most of them. Twenty-seven proteins were subjected to Edman microsequencing. N-terminal sequences of 12/27 proteins were determined, whereas internal sequences of 6/27 proteins were obtained following in situ proteolysis. Comparison between the experimental data and those reported in the SWISS-PROT database revealed some differences between genotypic and phenotypic sequences. These are indicative of the changes a protein can undergo with respect to the primary structure coded by the genomic DNA. Our results highlight the need to complement genomic analysis with detailed proteomics in order to refine the vast amount of information provided by DNA sequencing and to find an exact correlation between genome and proteome.     

Alpha‐Helical Fragaceatoxin C Nanopore Engineered for Double‐Stranded and Single‐Stranded Nucleic Acid Analysis

Nanopores are used in single‐molecule DNA analysis and sequencing. Herein, we show that Fragaceatoxin C (FraC), an α‐helical pore‐forming toxin from an actinoporin protein family, can be reconstituted in sphingomyelin‐free standard planar lipid bilayers. We engineered FraC for DNA analysis and show that the funnel‐shaped geometry allows tight wrapping around single‐stranded DNA (ssDNA), resolving between homopolymeric C, T, and A polynucleotide stretches. Remarkably, despite the 1.2 nm internal constriction of FraC, double‐stranded DNA (dsDNA) can translocate through the nanopore at high applied potentials, presumably through the deformation of the α‐helical transmembrane region of the pore. Therefore, FraC nanopores might be used in DNA sequencing and dsDNA analysis.     

Usefulness of microchip electrophoresis for the analysis of mitochondrial DNA in forensic and ancient DNA studies

We evaluate the usefulness of a commercially available microchip CE (MCE) device in different genetic identification studies performed with mitochondrial DNA (mtDNA) targets, including the haplotype analysis of HVR1 and HVR2 and the study of interspecies diversity of cytochrome b (Cyt b) and 16S ribosomal RNA (16S rRNA) mitochondrial genes in forensic and ancient DNA samples. The MCE commercial system tested in this study proved to be a fast and sensitive detection method of length heteroplasmy in cytosine stretches produced by 16 189T>C transitions in HVR1 and by 309.1 and 309.2 C-insertions in HVR2. Moreover, the quantitative analysis of PCR amplicons performed by LIF allowed normalizing the amplicon input in the sequencing reactions, improving the overall quality of sequence data. These quantitative data in combination with the quantification of genomic mtDNA by real-time PCR has been successfully used to evaluate the PCR efficiency and detection limit of full sequencing methods of different mtDNA targets. The quantification of amplicons also provided a method for the rapid evaluation of PCR efficiency of multiplex-PCR versus singleplex-PCR to amplify short HV1 amplicons (around 100 bp) from severely degraded ancient DNA samples. The combination of human-specific (Cyt b) and universal (16S rRNA) mtDNA primer sets in a single PCR reaction followed by MCE detection offers a very rapid and simple screening test to differentiate between human and nonhuman hair forensic samples. This method was also very efficient with degraded DNA templates from forensic hair and bone samples, because of its applicability to detect small amplicon sizes. Future possibilities of MCE in forensic DNA typing, including nuclear STRs and SNP profiling are suggested.     

Separation and analysis of DNA sequence reaction products by capillary gel electrophoresis

This paper demonstrates the potential of capillary gel electrophoresis with laser induced fluorescence detection as a tool for DNA sequence determination. Both synthetic oligonucleotides and single-stranded phage DNA were utilized as templates in the standard chain termination procedure. Primer molecules were tagged at the 5' end with the fluorescent dye, JOE. First, baseline resolution of a dA extended primer from 18 to 81 bases long, a total of 64 fragments, was observed. A second synthetic template was designed to yield alternating stretches of dA and dT extensions of the primer. Thirdly, the sequence reaction products from a synthetic oligonucleotide template containing all four bases was analyzed in four independent runs, one for each of the four base-specific reactions. In all cases, the expected number and patterns of peaks were observed by capillary gel electrophoretic analysis. Finally, separation of sequence reaction products generated with single-strand M13mp18 phage DNA as template exhibited baseline resolution of fragments differing in length by a single nucleotide and from 18 to greater than 330 bases total length.     

Escherichia coli single-stranded DNA-binding protein,a molecular tool for improved sequence quality in pyrosequencing

Pyrosequencing is a four-enzyme bioluminometric DNA sequencing technique based on a DNA sequencing by synthesis principle. Currently, the technique is limited to analysis of short DNA sequences exemplified by single-nucleotide polymorphism analysis. In order to expand the field for pyrosequencing, the read length needs to be improved and efforts have been made to purify reaction components as well as add single-stranded DNA-binding protein (SSB) to the pyrosequencing reaction. In this study, we have performed a systematic effort to analyze the effects of SSB by comparing the pyrosequencing result of 103 independent complementary DNA (cDNA) clones. More detailed information about the cause of low quality sequences on templates with different characteristics was achieved by thorough analysis of the pyrograms. Also, real-time biosensor analysis was performed on individual cDNA clones for investigation of primer annealing and SSB binding on these templates. Results from these studies indicate that templates with high performance in pyrosequencing without SSB possess efficient primer annealing and low SSB affinity. Alternative strategies to improve the performance in pyrosequencing by increasing the primer-annealing efficiency have also been evaluated.     

What is the future of electrophoresis in large-scale genomic sequencing?

Although a finished human genome reference sequence is now available, the ability to sequence large, complex genomes remains critically important for researchers in the biological sciences, and in particular, continued human genomic sequence determination will ultimately help to realize the promise of medical care tailored to an individual's unique genetic identity. Many new technologies are being developed to decrease the costs and to dramatically increase the data acquisition rate of such sequencing projects. These new sequencing approaches include Sanger reaction-based technologies that have electrophoresis as the final separation step as well as those that use completely novel, nonelectrophoretic methods to generate sequence data. In this review, we discuss the various advances in sequencing technologies and evaluate the current limitations of novel methods that currently preclude their complete acceptance in large-scale sequencing projects. Our primary goal is to analyze and predict the continuing role of electrophoresis in large-scale DNA sequencing, both in the near and longer term.     

Genomic Encryption of Digital Data Stored in Synthetic DNA

Today, we can read human genomes and store digital data robustly in synthetic DNA. Herein, we report a strategy to intertwine these two technologies to enable the secure storage of valuable information in synthetic DNA, protected with personalized keys. We show that genetic short tandem repeats (STRs) contain sufficient entropy to generate strong encryption keys, and that only one technology, DNA sequencing, is required to simultaneously read the key and the data. Using this approach, we experimentally generated 80 bit strong keys from human DNA, and used such a key to encrypt 17 kB of digital information stored in synthetic DNA. Finally, the decrypted information was recovered perfectly from a single massively parallel sequencing run.     

Denaturation mapping of Saccharomyces cerevisiae

Optical mapping of DNA provides large-scale genomic information that can be used to assemble contigs from next-generation sequencing, and to detect rearrangements between single cells. A recent optical mapping technique called denaturation mapping has the advantage of using physical principles rather than the action of enzymes to probe genomic structure. Denaturation mapping uses fluorescence microscopy to image the pattern of partial melting along a DNA molecule extended in a channel of cross-section 120 nm at the heart of a nanofluidic device. We used denaturation mapping to locate single DNA molecules on the yeast genome (12.1 Mbp) by comparing images to a computationally predicted map for the entire genome sequence. By locating 84 molecules we assembled an optical map of the yeast genome with > 50% coverage.     

De novo sequencing of highly modified therapeutic oligonucleotides by hydrophobic tag sequencing coupled with LC–MS

Correct sequences are prerequisite for quality control of therapeutic oligonucleotides. However, there is no definitive method available for determining sequences of highly modified therapeutic RNAs, and thereby, most of the oligonucleotides have been used clinically without direct sequence determination. In this study, we developed a novel sequencing method called ‘hydrophobic tag sequencing’. Highly modified oligonucleotides are sequenced by partially digesting oligonucleotides conjugated with a 5′‐hydrophobic tag, followed by liquid chromatography–mass spectrometry analysis. 5′‐Hydrophobic tag‐printed fragments (5′‐tag degradates) can be separated in order of their molecular masses from tag‐free oligonucleotides by reversed‐phase liquid chromatography. As models for the sequencing, the anti‐VEGF aptamer (Macugen) and the highly modified 38‐mer RNA sequences were analyzed under blind conditions. Most nucleotides were identified from the molecular weight of hydrophobic 5′‐tag degradates calculated from monoisotopic mass in simple full mass data. When monoisotopic mass could not be assigned, the nucleotide was estimated using the molecular weight of the most abundant mass. The sequences of Macugen and 38‐mer RNA perfectly matched the theoretical sequences. The hydrophobic tag sequencing worked well to obtain simple full mass data, resulting in accurate and clear sequencing. The present study provides for the first time a de novo sequencing technology for highly modified RNAs and contributes to quality control of therapeutic oligonucleotides. Copyright © 2016 John Wiley & Sons, Ltd.     

The application of magnetic bead hybridization for the recovery and STR amplification of degraded and inhibited forensic DNA

A common problem in the analysis of forensic DNA evidence is the presence of environmentally degraded and inhibited DNA. Such samples produce a variety of interpretational problems such as allele imbalance, allele dropout and sequence specific inhibition. In an attempt to develop methods to enhance the recovery of this type of evidence, magnetic bead hybridization has been applied to extract and preconcentrate DNA sequences containing short tandem repeat (STR) alleles of interest. In this work, genomic DNA was fragmented by heating, and sequences associated with STR alleles were selectively hybridized to allele-specific biotinylated probes. Each particular biotinylated probe-DNA complex was bound to streptavidin-coated magnetic beads using enabling enrichment of target DNA sequences. Experiments conducted using degraded DNA samples, as well as samples containing a large concentration of inhibitory substances, showed good specificity and recovery of missing alleles. Based on the favorable results obtained with these specific probes, this method should prove useful as a tool to improve the recovery of alleles from degraded and inhibited DNA samples.     

Electrochemical Detection of FAM134B Mutations in Oesophageal Cancer Based on DNA‐Gold Affinity Interactions

Inexpensive, simple and rapid DNA sensors capable of accurate and sensitive detection of cancer specific point mutations in DNA biomarkers are crucial for the routine screening of genetic mutations in cancer. Conventional approaches based on sequencing, mass spectroscopy, and fluorescence are highly effective, but they are tedious, slow and require labels and expensive equipment. Recent electrochemistry based approaches mostly rely on conventional DNA biosensing using recognition and transduction layers, and hence limited by the complicated steps of sensor fabrication associated with surface cleaning, self‐assembled monolayer formation, and target hybridization. Herein we report a relatively simple and inexpensive method for detecting point mutation in cancer by using the direct adsorption of purified DNA sequences onto an unmodified gold surface. The method relies on the base dependent affinity interaction of DNA with gold. Since the affinity interaction (adsorption) trend of DNA bases follows as adenine (A) > cytosine (C) > guanine (G)> thymine (T), two DNA sequences with different DNA base compositions (i. e., amplified mutated sequences will be distinctly different than its original sequence) will have different adsorption affinity towards gold. The amount of mutation sites on a DNA sequence is quantified by monitoring the electrochemical current as a function of the relative adsorption level of DNA samples onto a bare gold electrode. This method can successfully distinguish single point mutation in DNA from oesophageal cancer. We demonstrated the clinical utility of this approach by detecting different levels of mutations in tissue samples (n=9) taken from oesophageal cancer patients. Finally, the method was validated with High Resolution Melt (HRM) curve analysis and Sanger Sequencing.     

Quantitative experimental determination of primer-dimer formation risk by free-solution conjugate electrophoresis

DNA barcodes are short, unique ssDNA primers that "mark" individual biomolecules. To gain better understanding of biophysical parameters constraining primer-dimer formation between primers that incorporate barcode sequences, we have developed a capillary electrophoresis method that utilizes drag-tag-DNA conjugates to quantify dimerization risk between primer-barcode pairs. Results obtained with this unique free-solution conjugate electrophoresis approach are useful as quantitatively precise input data to parameterize computation models of dimerization risk. A set of fluorescently labeled, model primer-barcode conjugates were designed with complementary regions of differing lengths to quantify heterodimerization as a function of temperature. Primer-dimer cases comprised two 30-mer primers, one of which was covalently conjugated to a lab-made, chemically synthesized poly-N-methoxyethylglycine drag-tag, which reduced electrophoretic mobility of ssDNA to distinguish it from ds primer-dimers. The drag-tags also provided a shift in mobility for the dsDNA species, which allowed us to quantitate primer-dimer formation. In the experimental studies, pairs of oligonucleotide primer barcodes with fully or partially complementary sequences were annealed, and then separated by free-solution conjugate CE at different temperatures, to assess effects on primer-dimer formation. When less than 30 out of 30 base-pairs were bonded, dimerization was inversely correlated to temperature. Dimerization occurred when more than 15 consecutive base-pairs formed, yet non-consecutive base-pairs did not create stable dimers even when 20 out of 30 possible base-pairs bonded. The use of free-solution electrophoresis in combination with a peptoid drag-tag and different fluorophores enabled precise separation of short DNA fragments to establish a new mobility shift assay for detection of primer-dimer formation.     

Heterobifunctional modification of DNA for conjugation to solid surfaces

Many biosensors, DNA arrays, and next-generation DNA sequencing technologies need common methods for end modification of random DNA sequences generated from a sample of DNA. Surface immobilization of chemically modified DNA is often the first step in creating appropriate sensing platforms. We describe a simple technique for efficient heterobifunctional modification of arbitrary double-stranded DNA fragments with chosen chemical groups. The modification requires the use of short (10–20 base pairs) synthetic adaptors having desired terminal functional groups and installs known sequences, which can be used for hybridization of primers in the sequencing-by-synthesis approaches. The method, based on ligation under optimized conditions, is selective and provides high yields of the target heterobifunctional DNA product. An additional two-step procedure can be applied to select further for the desired bifunctionalized product using PCR amplification with a chemically modified primer. Both functional groups in the modified DNA are chemically active and can be used in surface immobilization of the DNA strands to create the surface of a biosensor or sequencing chip.     

Contaminant-induced current decline in capillary array electrophoresis

High-throughput capillary array electrophoresis (CAE) instruments for DNA sequencing suffer to varying degrees from read length degradation associated with electrophoretic current decline and inhibition or delay in the arrival of fragments at the detector. This effect is known to be associated with residual amounts of large, slow-moving fragments of template or genomic DNA carried through from sample preparation and sequencing reactions. Here, we investigate the creation and expansion of an ionic depletion region induced by overloading the capillary with low-mobility DNA fragments, and the effect of growth of this region on electrophoresis run failure. Slow-moving fragments are analytically and experimentally shown to reduce the ionic concentration of the downstream electrolyte. With injection of large fragments beyond a threshold quantity, the anode-side boundary of the nascent depletion region begins to propagate toward the anode at a rate faster than the contaminant DNA migration. Under such conditions, the depletion region expands, the capillary current declines dramatically, and the electrophoresis run yields a short read length or fails completely.     

Internal amino acid sequence analysis of proteins separated by gel electrophoresis after tryptic digestion in polyacrylamide matrix

Summary A method is described for obtaining peptide fragments for sequence analysis from microquantities of proteins separated by 1- or 2-dimensional polyacrylamide gel electrophoresis. After separation by electrophoresis, the proteins were stained with Coomassie Blue and excised. Proteolytic digestion with trypsin was performed directly in the polyacrylamide matrix. The resulting peptide fragments were eluted, separated by reversed phase HPLC, collected and sequenced in a gas phase sequencer. Excellent peptide recoveries allowed generation of extensive internal sequence information from picomole amounts of protein. The method thus overcomes the problem of obtaining amino acid sequence data from N-terminally blocked proteins and provides multiple, independent stretches of sequences that can be used to generate oligonucleotide probes for molecular cloning, to design synthetic peptides for inducing antibodies, and to search sequence databases for related proteins.     

Oligonucleotide fingerprinting of arrayed genomic DNA sequences using LNA-modified hybridization probes

Recently, we established a robust method for the detection of hybridization events using a DNA microarray deposited on a nanoporous membrane. Here, in a follow-up study, we demonstrate the performance of this approach on a larger set of LNA-modified oligoprobes and genomic DNA sequences. Twenty-six different LNA-modified 7-mer oligoprobes were hybridized to a set of 66 randomly selected human genomic DNA clones spotted on a nanoporous membrane slide. Subsequently, assay sensitivity analysis was performed using receiver operating characteristic (ROC) curves. Comparison of LNA-modified heptamers and DNA heptamers revealed that the LNA modification clearly improved sensitivity and specificity of hybridization experiment. Clustering analysis was applied in order to test practical performance of hybridization experiments with LNA-modified oligoprobes in recognizing similarity of genomic DNA sequences. Comparing the results with the theoretical sequence clusters, we conclude that the application of LNA-modified oligoprobes allows for reliable clustering of DNA sequences which reflects the underlying sequence homology. Our results show that LNA-modified oligoprobes can be used effectively to unravel sequence similarity of DNA sequences and thus, to characterize the content of unknown DNA libraries.