DNA Template Molecules Research Articles

Assessment of DNA quality for whole genome library preparation

In recent years, more sophisticated DNA technologies for genotyping have enabled considerable progress in various fields such as clinical genetics, archaeogenetics and forensic genetics. DNA samples previously rejected as too challenging to analyze due to low amounts of degraded DNA can now provide useful information. To increase the chances of success with the new methodologies, it is crucial to know the fragment size of the template DNA molecules, and whether the DNA in a sample is mostly single or double stranded. With this knowledge, an appropriate library preparation method can be chosen, and the DNA shearing parameters of the protocol can be adjusted to the DNA fragment size in the sample. In this study, we first developed and evaluated a user-friendly fluorometry-based protocol for estimation of DNA strandedness. We also evaluated different capillary electrophoresis methods for estimation of DNA fragmentation levels. Next, we applied the developed methodologies to a broad variety of DNA samples processed with different DNA extraction protocols. Our findings show that both the applied DNA extraction method and the sample type affect the DNA strandedness and fragmentation. The established protocols and the gained knowledge will be applicable for future sequencing-based high-density SNP genotyping in various fields.

Abstract LB097: Sensitive detection of central nervous system tumors using a sequencing based cerebrospinal fluid test

Abstract The current approach to diagnosing and monitoring tumors of the central nervous system relies almost exclusively on radiographic imaging and neurosurgical procedures. Cerebrospinal fluid (CSF) is appealing for diagnosis because it is already part of the standard of care for the diagnosis or management of several types of CNS disease, including cancer, and the tumor DNA is more highly concentrated inside the blood brain barrier than in plasma or other bodily fluids. Even though cytology is widely used, sensitivity remains low ranging from 2% to 50%, depending on cancer type. We report an analytic technique that efficiently introduces identical molecular barcodes to both strands of CSF template DNA molecules for the identification of cancer specific somatic mutations and copy number alterations. We apply this approach to 126 CSF samples obtained from individuals with known primary or metastatic tumors involving the brain and 36 CSF samples obtained from individuals with non-neoplastic neurological conditions such as multiple sclerosis and another neurologic conditions. We correctly detect 91% of cancers at a specificity of 94.5%. In cases (n=20) where standard of care cytology was available, cytology detected 25% of cancers while our molecular assay detected 90%. Our approach has the capacity to be used in combination with other clinical, radiologic, and laboratory-based data to inform the diagnosis and management of patients with suspected cancers of the brain. Positives n= Sensitivity GBM 42 46 91.3% Lymphoma 22 24 91.7% Medulloblastoma 14 16 87.5% Metastases 37 40 92.5% Citation Format: Christopher B. Douville, Chetan Bettegowda. Sensitive detection of central nervous system tumors using a sequencing based cerebrospinal fluid test [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB097.

On the causes, consequences, and avoidance of PCR duplicates: Towards a theory of library complexity.

Library preparation protocols for most sequencing technologies involve PCR amplification of the template DNA, which open the possibility that a given template DNA molecule is sequenced multiple times. Reads arising from this phenomenon, known as PCR duplicates, inflate the cost of sequencing and can jeopardize the reliability of affected experiments. Despite the pervasiveness of this artefact, our understanding of its causes and of its impact on downstream statistical analyses remains essentially empirical. Here, we develop a general quantitative model of amplification distortions in sequencing data sets, which we leverage to investigate the factors controlling the occurrence of PCR duplicates. We show that the PCR duplicate rate is determined primarily by the ratio between library complexity and sequencing depth, and that amplification noise (including in its dependence on the number of PCR cycles) only plays a secondary role for this artefact. We confirm our predictions using new and published RAD-seq libraries and provide a method to estimate library complexity and amplification noise in any data set containing PCR duplicates. We discuss how amplification-related artefacts impact downstream analyses, and in particular genotyping accuracy. The proposed framework unites the numerous observations made on PCR duplicates and will be useful to experimenters of all sequencing technologies where DNA availability is a concern.

Detection of rare mutations, copy number alterations, and methylation in the same template DNA molecules

The analysis of cell-free DNA (cfDNA) from plasma offers great promise for the earlier detection of cancer. At present, changes in DNA sequence, methylation, or copy number are the most sensitive ways to detect the presence of cancer. To further increase the sensitivity of such assays with limited amounts of sample, it would be useful to be able to evaluate the same template molecules for all these changes. Here, we report an approach, called MethylSaferSeqS, that achieves this goal, and can be applied to any standard library preparation method suitable for massively parallel sequencing. The innovative step was to copy both strands of each DNA-barcoded molecule with a primer that allows the subsequent separation of the original strands (retaining their 5-methylcytosine residues) from the copied strands (in which the 5-methylcytosine residues are replaced with unmodified cytosine residues). The epigenetic and genetic alterations present in the DNA molecules can then be obtained from the original and copied strands, respectively. We applied this approach to plasma from 265 individuals, including 198 with cancers of the pancreas, ovary, lung, and colon, and found the expected patterns of mutations, copy number alterations, and methylation. Furthermore, we could determine which original template DNA molecules were methylated and/or mutated. MethylSaferSeqS should be useful for addressing a variety of questions relating genetics and epigenetics.

Abstract 6609: Detection of rare mutations, copy number variation, and DNA methylation in the same template DNA molecules

Abstract The analysis of cell-free DNA (cfDNA) from plasma offers great promise for the earlier detection of cancer. At present, changes in DNA sequence, methylation, or copy number are the most sensitive ways to detect the presence of cancer. To increase the sensitivity of such assays, it would be useful to be able evaluate the same template molecules for all these changes. Here we report an approach, called MethylSaferSeqS, that achieves this goal, and can be applied to any standard library preparation method suitable for massively parallel sequencing. The innovative step was to copy both strands of each DNA-barcoded molecule with a primer that allows the subsequent separation of the original strands (retaining their 5-methylcytosine residues) from the copied strands (in which the 5-methylcytosine residues are replaced with unmodified cytosine residues). The original and copied strands are queried for epigenetic and genetic alterations, respectively. We applied this approach to plasma from 265 individuals, including 198 with cancers of the pancreas, ovary, lung or colon, and found the expected patterns of mutations, copy number alterations, and methylation. Furthermore, we could determine which original template DNA molecules were methylated and/or mutated. MethylSaferSeqS should be useful for addressing a variety of questions relating genetics and epigenetics in the future. Citation Format: Yuxuan Wang, Christopher Douville, Joshua Cohen, Chetan Bettegowda, Nick Papadopoulos, Ken Kinzler, Bert Vogelstein. Detection of rare mutations, copy number variation, and DNA methylation in the same template DNA molecules. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6609.

Isolation of E. coli RNA polymerase transcription elongation complexes by selective solid-phase photoreversible immobilization.

The ability to prepare defined transcription elongation complexes (TECs) is a fundamental tool for investigating the interplay between RNA polymerases (RNAPs) and nascent RNA. To facilitate the preparation of defined TECs that contain arbitrarily long and complex transcripts, we developed a procedure for isolating roadblocked E. coli TECs from an in vitro transcription reaction using solid-phase photoreversible immobilization. Our approach uses a modified DNA template that contains both a 5' photocleavable biotin tag and an internal biotin-TEG transcription stall site. Because the footprint of a TEC at the stall site sequesters the biotin-TEG tag, DNA template molecules that contain a TEC can be reversibly immobilized on streptavidin-coated magnetic beads by the 5' photocleavable biotin tag. In contrast, DNA template molecules that do not contain a TEC are retained on the beads because the biotin-TEG tag is exposed and can bind streptavidin. In this way, DNA template molecules that contain a TEC are gently separated from free DNA and DNA that contains non-productive transcription complexes. This procedure yields precisely positioned TECs that are >95% pure with >30% yield relative to the amount of input DNA template. The resulting complexes are >99% stable for at least 3h and can be used for biochemical investigations of nascent RNA structure and function in the context of E. coli RNAP. The procedure is likely generalizable to any RNAP that arrests at and sequesters the internal biotin-TEG stall site.

Consistent ultra-long DNA sequencing with automated slow pipetting

BackgroundOxford Nanopore Technologies’ instruments can sequence reads of great length. Long reads improve sequence assemblies by unambiguously spanning repetitive elements of the genome. Sequencing reads of significant length requires the preservation of long DNA template molecules through library preparation by pipetting reagents as slowly as possible to minimize shearing. This process is time-consuming and inconsistent at preserving read length as even small changes in volumetric flow rate can result in template shearing.ResultsWe have designed SNAILS (Slow Nucleic Acid Instrument for Long Sequences), a 3D-printable instrument that automates slow pipetting of reagents used in long read library preparation for Oxford Nanopore sequencing. Across six sequencing libraries, SNAILS preserved more reads exceeding 100 kilobases in length and increased its libraries’ average read length over manual slow pipetting.ConclusionsSNAILS is a low-cost, easily deployable solution for improving sequencing projects that require reads of significant length. By automating the slow pipetting of library preparation reagents, SNAILS increases the consistency and throughput of long read Nanopore sequencing.

Reducing noise and stutter in short tandem repeat loci with unique molecular identifiers

Unique molecular identifiers (UMIs) are a promising approach to contend with errors generated during PCR and massively parallel sequencing (MPS). With UMI technology, random molecular barcodes are ligated to template DNA molecules prior to PCR, allowing PCR and sequencing error to be tracked and corrected bioinformatically. UMIs have the potential to be particularly informative for the interpretation of short tandem repeats (STRs). Traditional MPS approaches may simply lead to the observation of alleles that are consistent with the hypotheses of stutter, while with UMIs stutter products bioinformatically may be re-associated with their parental alleles and subsequently removed. Herein, a bioinformatics pipeline named strumi is described that is designed for the analysis of STRs that are tagged with UMIs. Unlike other tools, strumi is an alignment-free machine learning driven algorithm that clusters individual MPS reads into UMI families, infers consensus super-reads that represent each family and provides an estimate the resulting haplotype’s accuracy. Super-reads, in turn, approximate independent measurements not of the PCR products, but of the original template molecules, both in terms of quantity and sequence identity. Provisional assessments show that naïve threshold-based approaches generate super-reads that are accurate (∼97 % haplotype accuracy, compared to ∼78 % when UMIs are not used), and the application of a more nuanced machine learning approach increases the accuracy to ∼99.5 % depending on the level of certainty desired. With these features, UMIs may greatly simplify probabilistic genotyping systems and reduce uncertainty. However, the ability to interpret alleles at trace levels also permits the interpretation, characterization and quantification of contamination as well as somatic variation (including somatic stutter), which may present newfound challenges.

Whole human blood DNA degradation associated with artificial ultraviolet and solar radiations as a function of exposure time

Laboratory investigations were conducted to evaluate the effect of ultraviolet radiation components and solar radiation exposure as a function of time on the degradation of whole human blood DNA from the standpoint of forensic analysis. Ten μL of whole human male blood samples were exposed to UV-A, UV-B, UV-C, and solar radiation at 20 min intervals up to 120 min. Allele frequencies of 16 short tandem repeat (STR) markers were monitored by employing current forensic typing DNA techniques. The STR markers were grouped into high, medium, and low molecular weight categories. Results revealed that even 20 min exposure to 4.89 eV UV-C photons (ʎ = 254 nm) with radiation intensity of 1200 μW/cm2 would degrade whole human male blood DNA samples significantly, making them unfit for human identification due to the breakdown of high molecular weight STRs. Exposure of blood samples to 4.11 eV UV-B photons (ʎ = 302 nm) with radiation intensity of 900 μW/cm2 resulted in complete degradation of high molecular weight STRs after 60 min. Partial breakdown of medium and low molecular weight STRs started after 80 min exposure. The degradation index (DI) values appear to show that the degradation of the DNA template molecule was relatively less in the low molecular weight DNA fragments as compared with high molecular weight DNA fragments. This finding indicates that genetic profiles obtained from whole human male blood exposed to this radiation for 60 min will give inconclusive results. Samples exposed up to 120 min to 3.40 eV UV-A photons (ʎ = 365 nm) and 3.10–3.94 eV photons of solar radiation did not appear to produce appreciable degradation in any of three molecular weight STRs in the whole human blood DNA samples.

Single-pass transcription by T7 RNA polymerase.

RNA molecules can be conveniently synthesized in vitro by the T7 RNA polymerase (T7 RNAP). In some experiments, such as cotranscriptional biochemical analyses, continuous synthesis of RNA is not desired. Here, we propose a method for a single-pass transcription that yields a single transcript per template DNA molecule using the T7 RNAP system. We hypothesized that stalling the polymerase downstream from the promoter region and subsequent cleavage of the promoter by a restriction enzyme (to prevent promoter binding by another polymerase) would allow synchronized production of a single transcript per template. The single-pass transcription was verified in two different scenarios: a short self-cleaving ribozyme and a long mRNA. The results show that a controlled single-pass transcription using T7 RNAP allows precise measurement of cotranscriptional ribozyme activity, and this approach will facilitate the study of other kinetic events.

CATS: Cas9-assisted tag switching. A high-throughput method for exchanging genomic peptide tags in yeast

BackgroundThe creation of arrays of yeast strains each encoding a different protein with constant tags is a powerful method for understanding how genes and their proteins control cell function. As genetic tools become more sophisticated there is a need to create custom libraries encoding proteins fused with specialised tags to query gene function. These include protein tags that enable a multitude of added functionality, such as conditional degradation, fluorescent labelling, relocalization or activation and also DNA and RNA tags that enable barcoding of genes or their mRNA products. Tools for making new libraries or modifying existing ones are becoming available, but are often limited by the number of strains they can be realistically applied to or by the need for a particular starting library.ResultsWe present a new recombination-based method, CATS – Cas9-Assisted Tag Switching, that switches tags in any existing library of yeast strains. This method employs the reprogrammable RNA guided nuclease, Cas9, to both introduce endogenous double strand breaks into the genome as well as liberating a linear DNA template molecule from a plasmid. It exploits the relatively high efficiency of homologous recombination in budding yeast compared with non-homologous end joining.ConclusionsThe method takes less than 2 weeks, is cost effective and can simultaneously introduce multiple genetic changes, thus providing a rapid, genome-wide approach to genetic modification.

Abstract 441: The stochastic nature of errors in next-generation sequencing of circulating cell-free DNA

Abstract Purpose: Challenges with distinguishing circulating tumor DNA from next-generation sequencing (NGS) artifacts limits variant searches to established solid tumor mutations. Identifying the source(s) of errors associated with the NGS analytics of circulating cell-free DNA (ccfDNA) would enable the determination of an optimal strategy for eliminating noise and broaden ccfDNA clinical applications. Methods: Buffy coat DNA and ccfDNA were isolated from seven healthy adults. For each participant, a single buffy coat DNA library was generated using duplex adapters (dual unique molecular identifiers [UMIs], dual index), while two ccfDNA libraries were separately produced - one library with singleton adapters (single UMI, single index) and one library with duplex adapters. The assignment of a UMI to each template DNA molecule prior to library formation reduces false positives. A family is a set of DNA amplicons (PCR duplicates) with the same UMI. Representing a family with a single consensus sequence reduces PCR errors and sequencing artifacts. Duplex adapters have been developed to abrogate the early PCR errors that beset singleton adapters. Results: The error rate using duplex adapters was significantly lower by 26.4±5.9% (P < 0.001) compared to singleton adapters at family size ≥2, where family size is defined as the number of PCR duplicates that yield a single consensus sequence. Due to the persistence of noise in both singleton and duplex adapters even at large family sizes (i.e., ≥10), we explored potential sources of the residual error. Noise in ccfDNA due to effects from clonal hematopoiesis of indeterminate potential (CHIP) accounted for <4% of error. Removing locations with errors present in all seven samples (i.e., highly patterned error likely due to regions difficult to sequence, align, or both) reduced noise in the duplex and singleton adapters by 18.7±5.1% (P < 0.001) and 14.5±3.1% (P < 0.001), respectively. Finally, we explored the effects of stochastic noise as a source of error. A complete replicate with duplex adapters was generated beginning from the source ccfDNA and library preparation. Using replicate data, the error rate was reduced by an additional 59.9±4.3% (P < 0.001) for the duplex adapters at family size ≥2. Using duplex adapters, accounting for CHIP artifacts, removing locations with highly patterned errors, and including replicate data reduced error by 85.4±4.6% (P < 0.001) compared to the error rate for singleton adapters at family size ≥2. Error continued to decline with each family size increment. Conclusion: Early stochastic PCR errors are a principal source of NGS noise that persist despite duplex molecular barcoding and after removal of patterned errors. Replicates are necessary to eliminate noise and their use in NGS analytics may broaden ccfDNA applications particularly in pre-metastatic and recurrent solid-tumor malignancies by enabling untargeted variant investigations. Citation Format: Hunter R. Underhill, Preetida J. Bhetariya, Sabine Hellwig, David A. Nix, Carrie L. Fuertes, Gabor T. Marth, Mary P. Bronner. The stochastic nature of errors in next-generation sequencing of circulating cell-free DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 441.

Uncoupling of sgRNAs from their associated barcodes during PCR amplification of combinatorial CRISPR screens.

Many implementations of pooled screens in mammalian cells rely on linking an element of interest to a barcode, with the latter subsequently quantitated by next generation sequencing. However, substantial uncoupling between these paired elements during lentiviral production has been reported, especially as the distance between elements increases. We detail that PCR amplification is another major source of uncoupling, and becomes more pronounced with increased amounts of DNA template molecules and PCR cycles. To lessen uncoupling in systems that use paired elements for detection, we recommend minimizing the distance between elements, using low and equal template DNA inputs for plasmid and genomic DNA during PCR, and minimizing the number of PCR cycles. We also present a vector design for conducting combinatorial CRISPR screens that enables accurate barcode-based detection with a single short sequencing read and minimal uncoupling.

Applications of Probe Capture Enrichment Next Generation Sequencing for Whole Mitochondrial Genome and 426 Nuclear SNPs for Forensically Challenging Samples.

The application of next generation sequencing (NGS) for the analysis of mitochondrial (mt) DNA, short tandem repeats (STRs), and single nucleotide polymorphism (SNPs) has demonstrated great promise for challenging forensic specimens, such as degraded, limited, and mixed samples. Target enrichment using probe capture rather than PCR amplification offers advantages for analysis of degraded DNA since two intact PCR primer sites in the template DNA molecule are not required. Furthermore, NGS software programs can help remove PCR duplicates to determine initial template copy numbers of a shotgun library. Moreover, the same shotgun library prepared from a limited DNA source can be enriched for mtDNA as well as nuclear markers by hybrid capture with the relevant probe panels. Here, we demonstrate the use of this strategy in the analysis of limited and mock degraded samples using our custom probe capture panels for massively parallel sequencing of the whole mtgenome and 426 SNP markers. We also applied the mtgenome capture panel in a mixed sample and analyzed using both phylogenetic and variant frequency based bioinformatics tools to resolve the minor and major contributors. Finally, the results obtained on individual telogen hairs demonstrate the potential of probe capture NGS analysis for both mtDNA and nuclear SNPs for challenging forensic specimens.

DNA multi-bit non-volatile memory and bit-shifting operations using addressable electrode arrays and electric field-induced hybridization

DNA has been employed to either store digital information or to perform parallel molecular computing. Relatively unexplored is the ability to combine DNA-based memory and logical operations in a single platform. Here, we show a DNA tri-level cell non-volatile memory system capable of parallel random-access writing of memory and bit shifting operations. A microchip with an array of individually addressable electrodes was employed to enable random access of the memory cells using electric fields. Three segments on a DNA template molecule were used to encode three data bits. Rapid writing of data bits was enabled by electric field-induced hybridization of fluorescently labeled complementary probes and the data bits were read by fluorescence imaging. We demonstrated the rapid parallel writing and reading of 8 (23) combinations of 3-bit memory data and bit shifting operations by electric field-induced strand displacement. Our system may find potential applications in DNA-based memory and computations.

Synthetic Cells Synthesize Therapeutic Proteins inside Tumors.

Synthetic cells, artificial cell-like particles, capable of autonomously synthesizing RNA and proteins based on a DNA template, are emerging platforms for studying cellular functions and for revealing the origins-of-life. Here, it is shown for the first time that artificial lipid-based vesicles, containing the molecular machinery necessary for transcription and translation, can be used to synthesize anticancer proteins inside tumors. The synthetic cells are engineered as stand-alone systems, sourcing nutrients from their biological microenvironment to trigger protein synthesis. When pre-loaded with template DNA, amino acids and energy-supplying molecules, up to 2 × 107 copies of green fluorescent protein are synthesized in each synthetic cell. A variety of proteins, having molecular weights reaching 66 kDa and with diagnostic and therapeutic activities, are synthesized inside the particles. Incubating synthetic cells, encoded to secrete Pseudomonas exotoxin A (PE) with 4T1 breast cancer cells in culture, resulted in killing of most of the malignant cells. In mice bearing 4T1 tumors, histological evaluation of the tumor tissue after a local injection of PE-producing particles indicates robust apoptosis. Synthetic cells are new platforms for synthesizing therapeutic proteins on-demand in diseased tissues.

Biochemistry: Complex assistance for DNA invasion.

Repair of broken DNA is vital for genome stability and to prevent the development of cancer. Research shows how the tumour-suppressor protein BRCA1 promotes a DNA-repair pathway called homologous recombination. See Article p.360 Two of the hereditary breast cancer susceptibility genes (BRCAs) act during the initial stages of recombinational DNA repair. BRCA1, together with BARD1, helps to form the single-stranded DNA that is then bound by another complex, BRCA2–PALB2, which facilitates loading of the central DNA strand exchange factor, RAD51. Patrick Sung and colleagues now show that BRCA1–BARD1 can also directly interact with RAD51 and stimulate the formation of the synaptic complex—a crucial intermediate that aligns the damaged and repair template DNA molecules. Because cancer cells depend on functioning DNA repair to thrive, targeting these factors may provide therapeutic value.

Multiple pathogen biomarker detection using an encoded bead array in droplet PCR

We present a droplet PCR workflow for detection of multiple pathogen DNA biomarkers using fluorescent color-coded Luminex® beads. This strategy enables encoding of multiple singleplex droplet PCRs using a commercially available bead set of several hundred distinguishable fluorescence codes. This workflow provides scalability beyond the limited number offered by fluorescent detection probes such as TaqMan probes, commonly used in current multiplex droplet PCRs. The workflow was validated for three different Luminex bead sets coupled to target specific capture oligos to detect hybridization of three microorganisms infecting poultry: avian influenza, infectious laryngotracheitis virus and Campylobacter jejuni. In this assay, the target DNA was amplified with fluorescently labeled primers by PCR in parallel in monodisperse picoliter droplets, to avoid amplification bias. The color codes of the Luminex detection beads allowed concurrent and accurate classification of the different bead sets used in this assay. The hybridization assay detected target DNA of all three microorganisms with high specificity, from samples with average target concentration of a single DNA template molecule per droplet. This workflow demonstrates the possibility of increasing the droplet PCR assay detection panel to detect large numbers of targets in parallel, utilizing the scalability offered by the color-coded Luminex detection beads.

Simple multiplexed PCR-based barcoding of DNA for ultrasensitive mutation detection by next-generation sequencing.

Detection of extremely rare variant alleles within a complex mixture of DNA molecules is becoming increasingly relevant in many areas of clinical and basic research, such as the detection of circulating tumor DNA in the plasma of cancer patients. Barcoding of DNA template molecules early in next-generation sequencing (NGS) library construction provides a way to identify and bioinformatically remove polymerase errors that otherwise make detection of these rare variants very difficult. Several barcoding strategies have been reported, but all require long and complex library preparation protocols. Simple, multiplexed, PCR-based barcoding of DNA for sensitive mutation detection using sequencing (SiMSen-seq) was developed to generate targeted barcoded libraries with minimal DNA input, flexible target selection and a very simple, short (∼4 h) library construction protocol. The protocol comprises a three-cycle barcoding PCR step followed directly by adaptor PCR to generate the library and then bead purification before sequencing. Thus, SiMSen-seq allows detection of variant alleles at <0.1% frequency with easy customization of library content (from 1 to 40+ PCR amplicons) and a protocol that can be implemented in any molecular biology laboratory. Here, we provide a detailed protocol for assay development and describe software to process the barcoded sequence reads.

Non-Homologous End Joining and Homology Directed DNA Repair Frequency of Double-Stranded Breaks Introduced by Genome Editing Reagents.

Genome editing using transcription-activator like effector nucleases or RNA guided nucleases allows one to precisely engineer desired changes within a given target sequence. The genome editing reagents introduce double stranded breaks (DSBs) at the target site which can then undergo DNA repair by non-homologous end joining (NHEJ) or homology directed recombination (HDR) when a template DNA molecule is available. NHEJ repair results in indel mutations at the target site. As PCR amplified products from mutant target regions are likely to exhibit different melting profiles than PCR products amplified from wild type target region, we designed a high resolution melting analysis (HRMA) for rapid identification of efficient genome editing reagents. We also designed TaqMan assays using probes situated across the cut site to discriminate wild type from mutant sequences present after genome editing. The experiments revealed that the sensitivity of the assays to detect NHEJ-mediated DNA repair could be enhanced by selection of transfected cells to reduce the contribution of unmodified genomic DNA from untransfected cells to the DNA melting profile. The presence of donor template DNA lacking the target sequence at the time of genome editing further enhanced the sensitivity of the assays for detection of mutant DNA molecules by excluding the wild-type sequences modified by HDR. A second TaqMan probe that bound to an adjacent site, outside of the primary target cut site, was used to directly determine the contribution of HDR to DNA repair in the presence of the donor template sequence. The TaqMan qPCR assay, designed to measure the contribution of NHEJ and HDR in DNA repair, corroborated the results from HRMA. The data indicated that genome editing reagents can produce DSBs at high efficiency in HEK293T cells but a significant proportion of these are likely masked by reversion to wild type as a result of HDR. Supplying a donor plasmid to provide a template for HDR (that eliminates a PCR amplifiable target) revealed these cryptic DSBs and facilitated the determination of the true efficacy of genome editing reagents. The results indicated that in HEK293T cells, approximately 40% of the DSBs introduced by genome editing, were available for participation in HDR.