Fiona J. Stewart

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.

Comprehensive nucleosome mapping of the human genome in cancer progression.

Altered chromatin structure is a hallmark of cancer, and inappropriate regulation of chromatin structure may represent the origin of transformation. Important studies have mapped human nucleosome distributions genome wide, but the role of chromatin structure in cancer progression has not been addressed. We developed a MNase-Transcription Start Site Sequence Capture method (mTSS-seq) to map the nucleosome distribution at human transcription start sites genome-wide in primary human lung and colon adenocarcinoma tissue. Here, we confirm that nucleosome redistribution is an early, widespread event in lung (LAC) and colon (CRC) adenocarcinoma. These altered nucleosome architectures are consistent between LAC and CRC patient samples indicating that they may serve as important early adenocarcinoma markers. We demonstrate that the nucleosome alterations are driven by the underlying DNA sequence and potentiate transcription factor binding. We conclude that DNA-directed nucleosome redistributions are widespread early in cancer progression. We have proposed an entirely new hierarchical model for chromatin-mediated genome regulation.

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 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

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.

Abstract 3628: Improving sequencing quality of libraries prepared from FFPE DNA

Targeted cancer therapy based on genomic alterations can be remarkably effective, and has made significant strides with the recent advances in next-generation sequencing (NGS) technology. Although samples of blood and other bodily fluids are being actively explored for early disease diagnosis and treatment monitoring, DNA isolated from FFPE samples is currently the main source for NGS-based cancer profiling in clinical settings. Unfortunately, sequencing DNA from FFPE samples is challenging due to limited quantities and poor quality, a result of DNA damage incurred during fixation and storage. Artifacts associated with FFPE DNA have limited the mutation detection sensitivity to ≥ 5% mutant allele frequency (Frampton et al, Nature Biotechnology 2013), which would unfortunately leave many low-abundance genetic variants of clinical significance undetected. For example, clinical resistance-causing KIT and EGFR mutations can be present in tumors at levels In this study, we investigated the effects of DNA repair and different sample handling workflows on sequencing quality of libraries prepared from FFPE samples. Careful analysis of sequencing data showed that base calling qualities for all 4 bases are improved, and aberrant G:C to A:T mutations were significantly reduced upon DNA repair. Because the large majority of mutations encountered in human tumors are G:C to A:T mutations (Greenman, C. Nature 2007), we expect that lowering the damage induced background noise of FFPE DNA would allow more reliable detection of clinically important, actionable mutations at lower abundance. In addition, we observed specific sequencing artifacts associated with the method of handling FFPE samples and have since identified effective measurements to avoid such artifacts. We expect that these improvements in sequencing quality of FFPE samples would ultimately enable more sensitive and robust detection of many low level genetic variations in clinically and biologically relevant cancer genes. Citation Format: pingfang liu, Lixin Chen, Laurence Ettwiller, Christine Sumner, Fiona J. Stewart, Eileen T. Dimalanta, Theodore B. Davis, Evans C. Thomas. Improving sequencing quality of libraries prepared from FFPE DNA. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3628.

Abstract 3998: Sequencing the B-cell and T-cell repertoire

Immune sequencing allows for the study of complex immunological diseases by sequencing millions of V(D)J combinations from B-cell antibody and T-cell receptors. The popularity of this technique has increased due to recent throughput and read length improvements in next-generation sequencing technologies. However, structural and sequence complexities of antibody genes have made reliable targeting approaches challenging. We have developed and optimized a method for accurate sequencing of full-length immune gene repertoires of B-cells and T-cells. The method uses a unique barcoding scheme specifically designed to tag every mRNA molecule with a unique identifier (UID) so that all PCR copies of each mRNA fragment can be collapsed into a single consensus sequence. This makes the assay extremely accurate, by resolving PCR bias and sequencing errors as well as allowing quantitative digital molecule counting. Immune sequencing libraries were generated from total RNA extracted from Peripheral Blood Mononuclear Cells in duplicate from a single patient. The use of UIDs enabled absolute quantification of starting RNA molecules present in the original sample and therefore accurate ranking of the antibody clone abundance, by avoiding the bias incorporated by PCR or sequencing when total reads only were measured. Using the same sequencing method, tumor samples were analyzed for abundance of expanded clones via grouping clones by V gene, J gene and CDR3 similarity and ranking by mRNA abundance. Additionally, the use of isotype-specific primers (IgM, IgD, IgG, IgA and IgE) enabled measurement of the heavy chain isotype proportions within the samples. Further, alignment of full-length heavy chain antibody sequences generated using this method to germline genes from reference databases enabled quantitation of the mutation level of each antibody sequence, thereby providing information on the overall maturity and mutational profile of the sample repertoire. Citation Format: Fiona J. Stewart, Mehmet Karaca, Kris Adams, Chris Clouser, Bonny Patel, Sonia Timberlake, William Donahue, Lynne Apone, Salvatore Russello, Eileen T. Dimalanta, Theodore B. Davis, Francois Vigneault. Sequencing the B-cell and T-cell repertoire. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3998.

Abstract 3186: Comparative analysis of different total RNA sequencing approaches

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, ILnnInitial transcriptomic studies largely relied on hybridization-based microarray technologies and offered a limited ability to fully catalogue and quantify the diverse RNA molecules that are expressed from genomes over wide ranges of levels. Massively parallel cDNA sequencing, or Total RNA-Seq, has allowed many advances in the characterization and quantification of transcriptomes; an offer a new appreciation for the complexity of the Transcriptome, encompassing a multitude of previously unknown coding and non-coding RNA species, particularly small RNAs, including micro RNAs. This work investigates the performance of different strategies for Total RNA-Seq using multiple next generation sequencing platforms. Standard RNA-sequencing approaches generally require double-stranded cDNA Synthesis, which erases RNA strand information. Synthesis of a randomly primed double-stranded cDNA followed by addition of adaptors for next-generation sequencing leads to the loss of information about which strand was present in the original mRNA template. The polarity of the transcript is important for correct annotation of novel genes, identification of antisense transcripts with potential regulatory roles, and for correct determination of gene expression levels in the presence of antisense transcripts. Here, we examine the performance of strand-specific RNA libraries made by direct ligation of adaptor on to the RNA. We analyze the effect of different RNA fragmentation methods (divalent cations plus heat versus enzymatic fragmentation) and we provide a comparative data analysis (library complexity, continuity of gene coverage, strand specificity and 3′and 5′-end bias analysis). Identification and analysis of small RNA by deep sequencing requires preparation of a di-tagged cDNA library, which leads to adaptor-dimer formation that strongly contaminates the library. We have developed a novel method to generate di-tagged small RNA libraries free of adapter-dimer contamination without introducing any additional enzymatic steps or gel purifications. This method has optimized the 3′adaptor-ligation reaction to recover and to increase representation of the 2′-O-modified RNAs present in a biological sample. To reduce cost and increase sample throughput we have developed a barcode strategy to tag samples during library construction. The multiplexed libraries can then be pooled together before size selection, reducing the number of steps in the workflow. This technique reduces bias by ligation, increases representation of modified small RNAs and simplifies workflow during library construction for small RNA analysis and discovery.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3186. doi:1538-7445.AM2012-3186

A fast solution to NGS library preparation with low nanogram DNA input.

Next-generation sequencing (NGS) has significantly impacted human genetics, enabling a comprehensive characterization of human genome as well as better understanding of many genomic abnormalities. By delivering massive DNA sequences at unprecedented speed and cost, NGS promises to make personalized medicine a reality in the foreseeable future. To date, library construction with clinical samples has been a challenge, primarily due to the limited quantities of sample DNA available. To overcome this challenge, we have developed a fast library preparation method using novel NEBNext reagents and adaptors, including a new DNA polymerase that has been optimized to minimize GC bias. This method enables library construction from an amount of DNA as low as 5 ng, and can be used for both intact and fragmented DNA. Moreover, the workflow is compatible with multiple NGS platforms.

Comparative analysis of strand-specific RNA sequencing approaches

Background Standard RNA sequencing approaches generally require double-stranded cDNA synthesis, which erases RNA strand information. Synthesis of a randomly primed double-stranded cDNA followed by addition of adaptors for next-generation sequencing leads to the loss of information about which strand was present in the original mRNA template. The polarity of the transcript is important for correct annotation of novel genes, identification of antisense transcripts with potential regulatory roles, and for correct determination of gene expression levels in the presence of antisense transcripts. Different strand-specific RNA-seq approaches have been developed to preserve information about strand polarity with different level of performances. Material and methods Using Illumina Deep Sequencing Technology, this work investigates the performance of two different directional RNA-Seq (strand-specific RNA-seq) strategies. One is based on direct ligation of adaptors to the RNA ends and the other is based on the labeling and excision of the second strand cDNA. The RNA-seq workflows present in this work have been improved over current more laborious RNA-seq methods. Their low RNA input and streamlined workflows make them compatible with high throughput and automation. We also analyze the effect of different RNA fragmentation methods (divalent cations plus heat versus enzymatic fragmentation). Results We will provide a comparative full data analysis of different strand-specific RNA methods (library performance, complexity, continuity of gene coverage, strand specificity, rRNA background). Conclusions Our results show improved methods for high-quality strand-specific RNA-seq library construction amenable to large-scale library construction and automation.