Theodore B. Davis

The MspJI family of modification-dependent restriction endonucleases for epigenetic studies

MspJI is a novel modification-dependent restriction endonuclease that cleaves at a fixed distance away from the modification site. Here, we present the biochemical characterization of several MspJI homologs, including FspEI, LpnPI, AspBHI, RlaI, and SgrTI. All of the enzymes specifically recognize cytosine C5 modification (methylation or hydroxymethylation) in DNA and cleave at a constant distance (N12/N16) away from the modified cytosine. Each displays its own sequence context preference, favoring different nucleotides flanking the modified cytosine. By cleaving on both sides of fully modified CpG sites, they allow the extraction of 32-base long fragments around the modified sites from the genomic DNA. These enzymes provide powerful tools for direct interrogation of the epigenome. For example, we show that RlaI, an enzyme that prefers mCWG but not mCpG sites, generates digestion patterns that differ between plant and mammalian genomic DNA, highlighting the difference between their epigenomic patterns. In addition, we demonstrate that deep sequencing of the digested DNA fragments generated from these enzymes provides a feasible method to map the modified sites in the genome. Altogether, the MspJI family of enzymes represent appealing tools of choice for method development in DNA epigenetic studies.

A Method for Selectively Enriching Microbial DNA from Contaminating Vertebrate Host DNA

DNA samples derived from vertebrate skin, bodily cavities and body fluids contain both host and microbial DNA; the latter often present as a minor component. Consequently, DNA sequencing of a microbiome sample frequently yields reads originating from the microbe(s) of interest, but with a vast excess of host genome-derived reads. In this study, we used a methyl-CpG binding domain (MBD) to separate methylated host DNA from microbial DNA based on differences in CpG methylation density. MBD fused to the Fc region of a human antibody (MBD-Fc) binds strongly to protein A paramagnetic beads, forming an effective one-step enrichment complex that was used to remove human or fish host DNA from bacterial and protistan DNA for subsequent sequencing and analysis. We report enrichment of DNA samples from human saliva, human blood, a mock malaria-infected blood sample and a black molly fish. When reads were mapped to reference genomes, sequence reads aligning to host genomes decreased 50-fold, while bacterial and Plasmodium DNA sequences reads increased 8–11.5-fold. The Shannon-Wiener diversity index was calculated for 149 bacterial species in saliva before and after enrichment. Unenriched saliva had an index of 4.72, while the enriched sample had an index of 4.80. The similarity of these indices demonstrates that bacterial species diversity and relative phylotype abundance remain conserved in enriched samples. Enrichment using the MBD-Fc method holds promise for targeted microbiome sequence analysis across a broad range of sample types.

High Sensitivity 5-hydroxymethylcytosine Detection in Balb/C Brain Tissue

DNA hydroxymethylation is a long known modification of DNA, but has recently become a focus in epigenetic research. Mammalian DNA is enzymatically modified at the 5(th) carbon position of cytosine (C) residues to 5-mC, predominately in the context of CpG dinucleotides. 5-mC is amenable to enzymatic oxidation to 5-hmC by the Tet family of enzymes, which are believed to be involved in development and disease. Currently, the biological role of 5-hmC is not fully understood, but is generating a lot of interest due to its potential as a biomarker. This is due to several groundbreaking studies identifying 5-hydroxymethylcytosine in mouse embryonic stem (ES) and neuronal cells. Research techniques, including bisulfite sequencing methods, are unable to easily distinguish between 5-mC and 5-hmC . A few protocols exist that can measure global amounts of 5-hydroxymethylcytosine in the genome, including liquid chromatography coupled with mass spectrometry analysis or thin layer chromatography of single nucleosides digested from genomic DNA. Antibodies that target 5-hydroxymethylcytosine also exist, which can be used for dot blot analysis, immunofluorescence, or precipitation of hydroxymethylated DNA, but these antibodies do not have single base resolution.In addition, resolution depends on the size of the immunoprecipitated DNA and for microarray experiments, depends on probe design. Since it is unknown exactly where 5-hydroxymethylcytosine exists in the genome or its role in epigenetic regulation, new techniques are required that can identify locus specific hydroxymethylation. The EpiMark 5-hmC and 5-mC Analysis Kit provides a solution for distinguishing between these two modifications at specific loci. The EpiMark 5-hmC and 5-mC Analysis Kit is a simple and robust method for the identification and quantitation of 5-methylcytosine and 5-hydroxymethylcytosine within a specific DNA locus. This enzymatic approach utilizes the differential methylation sensitivity of the isoschizomers MspI and HpaII in a simple 3-step protocol. Genomic DNA of interest is treated with T4-BGT, adding a glucose moeity to 5-hydroxymethylcytosine. This reaction is sequence-independent, therefore all 5-hmC will be glucosylated; unmodified or 5-mC containing DNA will not be affected. This glucosylation is then followed by restriction endonuclease digestion. MspI and HpaII recognize the same sequence (CCGG) but are sensitive to different methylation states. HpaII cleaves only a completely unmodified site: any modification (5-mC, 5-hmC or 5-ghmC) at either cytosine blocks cleavage. MspI recognizes and cleaves 5-mC and 5-hmC, but not 5-ghmC. The third part of the protocol is interrogation of the locus by PCR. As little as 20 ng of input DNA can be used. Amplification of the experimental (glucosylated and digested) and control (mock glucosylated and digested) target DNA with primers flanking a CCGG site of interest (100-200 bp) is performed. If the CpG site contains 5-hydroxymethylcytosine, a band is detected after glucosylation and digestion, but not in the non-glucosylated control reaction. Real time PCR will give an approximation of how much hydroxymethylcytosine is in this particular site. In this experiment, we will analyze the 5-hydroxymethylcytosine amount in a mouse Babl/C brain sample by end point PCR.

A λgt11 cDNA recombinant that encodes Dirofilaria immitis paramyosin

The cDNA synthesized from mRNA of Dirofilaria immitis female adult worms was cloned into the expression vector lambda gt11. Screening the library with a hyperimmune rabbit antiserum raised against adult worm homogenates yielded several antigen positive clones. One of these clones, lambda cDi2, was recognized by rabbit antisera raised against either D. immitis L-3, adult, Brugia malayi L-3 or Onchocerca volvulus adult worm antigen, as well as by antisera from humans naturally infected with O. volvulus or Wuchereria bancrofti. Affinity-purified anti-lambda cDi2 antibodies reacted with a 97-kDa protein on Western transfers of adult D. immitis antigen extracts that were reduced with beta-mercaptoethanol. The whole rabbit anti-D. immitis adult antiserum depleted of anti-lambda cDi2 antibodies exhibited decreased reactivity to this 97-kDa band. A monoclonal antibody (IA6) that specifically binds Schistosoma mansoni paramyosin also recognised a 97-kDa protein in D. immitis extracts upon Western transfer. The deduced amino acid sequence of partial DNA sequence from lambda cDi2 showed some similarity to nematode myosin, and gave a stretch of 82 amino acids that is 91.5% identical to Caenorhabditis elegans paramyosin: thus, lambda cDi2 encodes D. immitis paramyosin.

A Microbiome DNA Enrichment Method for Next‐Generation Sequencing Sample Preparation

“Microbiome” is used to describe the communities of microorganisms and their genes in a particular environment, including communities in association with a eukaryotic host or part of a host. One challenge in microbiome analysis concerns the presence of host DNA in samples. Removal of host DNA before sequencing results in greater sequence depth of the intended microbiome target population. This unit describes a novel method of microbial DNA enrichment in which methylated host DNA such as human genomic DNA is selectively bound and separated from microbial DNA before next‐generation sequencing (NGS) library construction. This microbiome enrichment technique yields a higher fraction of microbial sequencing reads and improved read quality resulting in a reduced cost of downstream data generation and analysis.

Abstract 1425: A multi-enzyme DNA repair mix improves library quality and sequencing accuracy in FFPE tumor samples

Next-generation sequencing (NGS) methods are used extensively to profile mutations present in diseased human tissues. These genomic approaches hold great promise for personalized medicine but sequencing accuracy is essential for proper patient diagnosis and determining a treatment plan. A common source of DNA for genomic profiling is formalin-fixed, paraffin-embedded (FFPE) tissue samples obtained from patient biopsy. FFPE DNA poses important challenges for preparing NGS libraries including low input amounts and poor DNA quality, resulting from extensive fixation- and storage-induced DNA damage. Additionally, these damage-induced sequencing artifacts raise the background level of mutations, making it difficult to discern true, low frequency, disease-causing variants from noise. We previously showed that a major fraction of somatic mutations described in publicly available datasets are due to such sequencing artifacts (Chen et al., Science 2017). Furthermore, we showed that enzymatic repair of DNA before library preparation improves the library quality and reduces background noise. We developed a second-generation DNA repair enzyme mix (V2) that efficiently repairs the most prevalent damage types found in FFPE DNA and further improves the quality and yield of NGS libraries. Additionally, we tested the efficacy of the V2 repair mix in improving sequencing accuracy for FFPE DNA samples obtained from different cancer tissues. We performed target enrichment on a panel of 151 cancer genes, deep sequenced, and performed variant analysis. For a subset of variants, we further validated our results using a droplet digital PCR (ddPCR) assay. Both methods showed that the V2 repair mix did not alter the overall frequency of variants identified, thus it did not introduce bias, but significantly improved the sequencing accuracy by reducing the number of false variant calls. Therefore, enzymatic repair is a critical first step in preparing FFPE DNA sequencing libraries, allowing more sensitive and robust detection of low frequency, disease variants. Citation Format: Pingfang Liu, Margaret Heider, Chen Song, Lixin Chen, Laurence Ettwiller, Lauren Higgins, Eileen Dimalanta, Theodore Davis, Thomas Evans. A multi-enzyme DNA repair mix improves library quality and sequencing accuracy in FFPE tumor samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1425.

Abstract 5365: Combining enzymatic DNA fragmentation with NGS library construction results in high quality, high yield libraries

The use of Next Generation Sequencing (NGS) data has been instrumental in advancing our understanding of human genetics, identifying the molecular events that contribute to human disease, and supporting drug development targeted towards precision medicine. Continued advancement relies on overcoming the limitations and bottlenecks associated with NGS. In this work, we have focused on NGS library preparation, where the requirement for expensive equipment and numerous steps can lead to sample loss, errors, and limited throughput. Specifically, we have developed a library construction method that integrates enzymatic DNA fragmentation into the workflow and combines fragmentation with end repair and dA-tailing in a single step. Integrating these reactions eliminates the need for costly equipment to shear DNA and reduces the number of sample transfers and losses. Adaptor ligation is also carried out in the same tube, after which a single cleanup step is performed. For low input samples, PCR amplification is performed prior to sequencing. This method is compatible with a broad range of DNA inputs and insert sizes. Libraries generated using this streamlined method with inputs ranging from 500 pg to 500 ng of intact DNA show no significant difference in coverage uniformity or sequence quality metrics, compared to libraries generated with mechanically sheared DNA. Similarly, libraries generated to contain insert sizes that range from 150bp to 1kb display no significant difference in sequence quality from each other or from those generated with mechanically sheared DNA. Finally, this streamlined method generates libraries of substantially higher yields than those generated using mechanically fragmented DNA, allowing the use of lower DNA inputs and fewer PCR cycles. The ability to generate high quality NGS libraries from intact DNA without the need for costly equipment and numerous cleanup or liquid transfer steps substantially reduces the time, cost and errors associated with library construction. In addition, these advances will enable greater use and adoption of NGS technologies in clinical and diagnostic settings. Citation Format: Fiona Stewart, Lynne Apone, Vaishnavi Panchapakesa, Karen Duggan, Timur Shtatland, Bradley Langhorst, John Murdoch, Christine Sumner, Christine Rozzi, Pingfang Liu, Keerthana Krishnan, Deyra Rodriguez, Joanna Bybee, Danielle Rivizzigno, Laurie Mazzola, Eileen Dimalanta, Theodore Davis. Combining enzymatic DNA fragmentation with NGS library construction results in high quality, high yield libraries [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5365. doi:10.1158/1538-7445.AM2017-5365

Abstract 5362: Targeting BRCA1 and BRCA2 with NEBNext Direct™

The screening and detection of germline BRCA1 and BRCA2 mutations are critical for the effective management of patients with breast or ovarian cancer and for the identification of individuals with a high risk of developing these cancers. In addition, somatic detection of pathogenic variants in BRCA1/2 can influence treatment decisions due to the susceptibility of tumors with BRCA mutations to PARP inhibitors. Here we introduce the NEBNext DirectTM BRCA enrichment panel for the interrogation of BRCA1 and BRCA2 by Illumina sequencing. NEBNext DirectTM is a novel, hybridization-based method to selectively enrich nucleic acid targets ranging from a single gene to several hundred genes. This approach includes features such as the incorporation of unique molecule identifiers (UMIs) and the ability to capture degraded DNA, enabling accurate detection of low-frequency variants from formalin-compromised DNA and other challenging sample types. We applied the NEBNext Direct BRCA1/2 panel to frozen tissue and achieved a high specificity for the BRCA targets (96% of the sequenced reads mapped to BRCA1 and BRCA2 and 80% of the sequenced bases were within the targeted regions). In addition, the resulting libraries were highly uniform in coverage, with 91% of the targeted bases covered at a value of at least 50% of the mean depth of coverage and 100% of the bases covered at 20% of the mean or greater. Some variability in the specificity was observed with formalin-fixed, paraffin embedded (FFPE) samples, and this effect was dependent on the quality of the FFPE DNA. The UMIs were used to identify PCR duplicates prior to variant calling, as well as for error correction, enabling the accurate detection of low-frequency variants. The library preparation for all samples was completed in one day, and the entire process of library preparation, sequencing on the Illumina Miseq, and data analysis was completed in a total of two days. In summary, we demonstrate that application of the NEBNext DirectTM method to the enrichment of BRCA1 and BRCA2 provides a tractable approach for the rapid and highly sensitive analysis of these cancer-associated genes. Citation Format: Scott M. Adams, Kruti M. Patel, Amy B. Emerman, Sarah K. Bowman, Charles D. Elfe, Noa Henig, Salvatore Russello, Andrew Barry, Theodore Davis, Cynthia L. Hendrickson. Targeting BRCA1 and BRCA2 with NEBNext Direct™ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5362. doi:10.1158/1538-7445.AM2017-5362

Abstract 5360: DNA repair increases sequencing accuracy without altering actual mutation frequency in clinical samples

Targeted cancer therapy based on genomic alterations can be remarkably effective. Currently, cancer genome profiling using next generation sequencing (NGS) is routinely applied in cancer care to guide personalized treatment. The accuracy of this profiling directly impacts therapeutic choices and the outcomes of patient care. We previously showed that false positive variants are abundant and can account for a major fraction of identified somatic variations in publicly available datasets (doi: http://dx.doi.org/10.1101/070334). These false positive variants show signs of mutagenic DNA damage. We further demonstrated that enzymatic DNA repair increases sequencing quality by lowering damage-induced background noise. Therefore, enzymatic DNA repair has the potential to improve sequencing accuracy, avoiding incorrect somatic variant calls and consequently reducing incorrect diagnostic conclusions. In this study, we investigated whether enzymatic DNA repair introduces any bias to NGS libraries using analysis by droplet digital PCR (ddPCR) and deep sequencing. DNA Reference Standards containing multiple common cancer mutations (Horizon Discovery, Inc.) were spiked into formalin-fixed paraffin-embedded (FFPE) DNA isolated from tumor samples from different tissue types at defined frequencies (0.5-10% quantified by ddPCR). Genotyping of the FFPE DNA ensured that they were free of any of the spiked-in mutations. After DNA repair and library preparation, mutation frequencies were quantified by ddPCR, and compared to the mutation levels in input DNA and control libraries without repair. Deep sequencing of 151 cancer genes including these spike-ins showed no difference in mutation frequency for the spiked-in mutations between the control and repair groups. However, the number of false positive variant calls was reduced in the repair group. Our data demonstrates that DNA repair significantly increases sequencing accuracy without altering the frequency of actual mutations in tumor samples. Citation Format: Pingfang Liu, Lixin Chen, Laurence Ettwiller, Eileen Dimalanta, Theodore B. Davis, Thomas C. Evans. DNA repair increases sequencing accuracy without altering actual mutation frequency in clinical samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5360. doi:10.1158/1538-7445.AM2017-5360

Selective Depletion of Abundant RNAs to Enable Transcriptome Analysis of Low‐Input and Highly Degraded Human RNA

Ribosomal RNAs (rRNAs) are extremely abundant, often constituting 80% to 90% of total RNA. Since rRNA sequences are often not of interest in genomic RNA sequencing experiments, rRNAs can be removed from the sample before the library preparation step, in order to prevent the majority of the library and the majority of sequencing reads from being rRNA. Removal of rRNA can be especially challenging for low quality and formalin‐fixed paraffin‐embedded (FFPE) RNA samples due to the fragmented nature of these RNA molecules. The NEBNext rRNA Depletion Kit (Human/Mouse/Rat) depletes both cytoplasmic (5 S rRNA, 5.8 S rRNA, 18 S rRNA, and 28 S rRNA) and mitochondrial rRNA (12 S rRNA and 16 S rRNA) from total RNA preparations from human, mouse, and rat samples. Due to the high similarity among mammalian rRNA sequences, it is likely that rRNA depletion can also be achieved for other mammals but has not been empirically tested. This product is compatible with both intact and degraded RNA (e.g., FFPE RNA). The resulting rRNA‐depleted RNA is suitable for RNA‐seq, random‐primed cDNA synthesis, or other downstream RNA analysis applications. Regardless of the quality or amount of input RNA, this method efficiently removes rRNA, while retaining non‐coding and other non‐poly(A) RNAs. The NEBNext rRNA Depletion Kit thus provides a more complete picture of the transcript repertoire than oligo d(T) poly(A) mRNA enrichment methods.