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Next Generation Sequencing (NGS) is rapidly becoming a powerful tool, by providing a high resolution and global view of the cancer genome mainly through whole genome, whole exome and transcriptome sequencing. This knowledge is important to support drug development targeted towards precision medicine. Obtaining high quality sequencing data begins with optimal sample preparation. DNA fragmentation is one of the critical first steps in the construction of high quality libraries for NGS, but current fragmentation methods create a bottleneck on library preparation throughput. We have developed an efficient, one tube fragmentation system that integrates enzyme-based DNA fragmentation with end-repair and dA-tailing in a single step, followed by adaptor ligation in the same tube. This streamlined method is compatible with a wide range of DNA samples and input amounts, resulting in reproducible and reliable fragment sizes. Genomic DNA (100pg to 500ng) with various GC content, was used in the construction of libraries, that were sequenced on an Illumina platform. For low input samples, PCR amplification was performed prior to sequencing. Libraries constructed using intact genomic DNA and this novel library preparation method produced substantially higher yields than standard methods and mechanically-fragmented DNA. Enzyme-based shearing increases library yield by reducing DNA damage and sample loss. High library yields also enable PCR-free workflows from as little as 50ng starting material. This method can be used for DNA extraction and library preparation from whole blood and dried blood samples.Sequencing quality of libraries generated with inputs ranging from 100pg to 500ng DNA show similar coverage uniformity and fragment size distribution, with minimal GC bias. We have developed an enzymatic DNA fragmentation kit that enables the construction of high quality libraries from a range of sample types and inputs with minimal sequence bias, high yields and low duplication rates. This method eliminates the need for expensive equipment to fragment DNA as well as numerous cleanup and liquid transfer steps thereby reducing the time, cost and errors associated with library construction. These advances in NGS technologies, supported by powerful bioinformatics tools, promises to revolutionize cancer research, diagnosis and therapy. Citation Format: Vaishnavi Panchapakesa, Lynne Apone, Karen Duggan, Bradley Langhorst, Chen Song, Pingfang Liu, Timur Shtatland, Christine Rozzi, Christine Sumner, Fiona Stewart, Eileen Dimalanta, Theodore Davis. Novel enzymatic DNA fragmentation integrated into library construction [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 3528.

The study of complex immunological diseases and tumor microenvironments has progressed through recent developments on sequencing of the immune repertoire. Using this approach, the interrogation of disease progression is facilitated through analysis of millions of V(D)J combinations from B cell antibodies (Igs) and T cell receptors (TCRs). One major challenge of immune repertoire sequencing is to accurately capture the structural and sequence complexities of antibodies and TCR genes. We have developed a method for accurate sequencing of full length immune gene repertoires of B cells and T cells. RNA extracted from tumor tissues containing tumor infiltrating lymphocytes, as well as matched peripheral blood mononuclear cells (PBMCs) were used to generate full length Ig and TCR libraries. Unique molecule index (UMI) was used to discretely barcode each mRNA molecule, enabling absolute quantitative ranking of antibody/TCR clone abundance. Full length immune repertoire sequencing facilitates detection of distinct and shared clones in tissue and blood samples, enabling identification of disease specific clones to evaluate immunotherapy effects. Highly expanded clones in tumor samples are also found in blood samples. RNA-seq libraries were also constructed from the same RNA for Ig and TCR libraries. The expression level of IGH/IGL/IGK and TRA/TRB in the immune sequencing libraries highly correlates with the RNA-seq data. In addition, both Ig and TCR libraries can be constructed in one tube to obtain a whole immune repertoire profile.Our immune repertoire sequencing approach allows accurate clonal determination for both Ig and TCR. This technique is applicable for investigation of lymphocytes infiltration of tumor microenvironments, tracing expanded B cell and T cells in blood samples, and monitoring of minimal residual disease. Citation Format: Chen Song, Pingfang Liu, Andrew Barry, Bradley W. Langhorst, Fiona J. Stewart, Salvatore Russello, Theodore B. Davis, Eileen T. Dimalanta. Immune repertoire sequencing reveals tumor microenvironment and tracks clonally expanded B cell and T cell in blood [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 4046.

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. © 2016 by John Wiley & Sons, Inc. Keywords: NGS; RNA depletion; sequencing; formalin-fixed paraffin-embedded; FFPE; RNA; rRNA; ribosomal RNA; library preparation; transcriptome; rRNA removal

The study of complex immunological diseases has progressed through recent developments that enable the sequencing of the immune repertoire. Using this approach, the interrogation of disease progression is facilitated through analysis of millions of V(D)J combinations from both B-cell antibodies and T-cell Receptors (TCR). One major challenge of immune repertoire sequencing is to capture the structural and sequence complexities of antibody and TCR genes. We have developed and optimized a method for accurate sequencing of full-length immune gene repertoires of B-cells and T-cells. RNA extracted from PBMCs were used to generate immune sequencing libraries in duplicate from a single patient. Using a unique barcoding scheme specifically designed to discretely barcode each mRNA molecule with a unique molecule index (UMI), PCR copies of each mRNA fragment can be collapsed into a single consensus sequence, improving accuracy through the resolution of PCR bias and sequencing errors. Utilization of the UMIs enabled absolute quantification of input RNA molecules and accurate ranking of the antibody/TCR clone abundance. This approach was also applied to the analysis of tumor samples for abundance of expanded clones through clustering based on V gene, J gene and CDR3 similarity and ranking by mRNA abundance. The use of isotype-specific primers (IgM, IgD, IgG, IgA and IgE) enabled measurement of the heavy chain isotype proportions within the samples. Full-length heavy chain antibody sequences generated using this method were aligned to germline genes from reference databases, enabling quantitation of the mutation level of each antibody sequence, providing information on the overall maturity and mutational profile of the sample repertoire. Additionally, this approach was capable of detecting minimal residual diseases by sensitive quantification of TCR rearrangement. This novel method allows for exhaustive somatic mutation profiling across complete V, D and J segments and full isotype information analysis. The information obtained enables the possibility for synthesis and expression of complete antibody chains and T-cell receptor chains for downstream immunological assays. Citation Format: Chen Song, Pingfang Liu, Andrew Barry, Eileen T. Dimalanta, Fiona J. Stewart, Salvatore Russello, Theodore B. Davis. B-cell and T-cell repertoire sequencing enables somatic hypermutation and minimal residual disease assessment [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 4675.

// Brooke R. Druliner 1,5 , Daniel Vera 1,6 , Ruth Johnson 2 , Xiaoyang Ruan 3 , Lynn M. Apone 4 , Eileen T. Dimalanta 4 , Fiona J. Stewart 4 , Lisa Boardman 5 and Jonathan H. Dennis 1,6,7 1 Department of Biological Science, Florida State University, Tallahassee, Florida, United States of America 2 Department of Laboratory Medicine and Experimental Pathology, Mayo Clinic, Rochester, Minnesota, United States of America 3 Biomedical Statistics and Informatics, Department of Health Sciences Research, Mayo Clinic College of Medicine, Rochester, Minnesota, United States of America 4 New England Biolabs Inc., Ipswich, Massachusetts, United States of America 5 Division of Gastroenterology and Hepatology, Department of Internal Medicine, Mayo Clinic, Rochester, Minnesota, United States of America 6 The Center for Genomics and Personalized Medicine, The Florida State University, Tallahassee, Florida, United States of America 7 Institute of Molecular Biophysics, The Florida State University, Tallahassee, Florida, United States of America Correspondence to: Jonathan H. Dennis, email: // Keywords : cancer, chromatin, nucleosome, MNase, whole genome, Chromosome Section Received : December 18, 2015 Accepted : December 21, 2015 Published : December 31, 2015 Abstract 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.

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

RNA-seq has become the most popular method for transcriptome analysis and is widely used to study gene expression, detect mutations, fusion transcripts, alternative splicing, and post-transcriptional modifications. It is becoming the method of choice to detect alterations in diseases, especially cancer, to provide insights on the various molecular pathways perturbed by changes in the transcriptome and study their implications. As RNA-seq is being adopted in molecular diagnostics and biomarker identifications, the need for good quality, reproducible library preparation methods using very low amounts of RNA input, or precious clinical samples, is increasing. To address these challenges, we have developed a strand-specific RNA-seq library preparation method that retains information about which strand of DNA is transcribed, from as low as 5 ng total RNA input. Strand specificity is important for correct annotation of genes, identification of antisense transcripts with potential regulatory roles, and accurate determination of gene expression levels in the presence of antisense transcripts. Enhanced sensitivity to detect transcripts with even coverage across their length offers a non-biased approach for accurate quantification of transcript levels. Methods: Enriched poly-A mRNA or ribo-depleted RNA (Universal Human Reference RNA) was used to make libraries with our strand specific library preparation method. Library quality and quantity were analyzed on an Agilent Bioanalyzer, pooled at equimolar ratio and sequenced on Illumina’s Nextseq 500. Paired end reads were mapped to a human reference genome (hg19) using Hisat2 and sequencing metrics were calculated using Picard's RNA-seq Metrics and RSeqC tools. Transcript abundance was measured using Salmon and the Ensembl GRCh38 CDS sequences. Results: Libraries prepared with our streamlined method using inputs that range from 5ng to 1ug show greater than 98% directionality at all input levels. GC content analysis, gene body coverage and gene expression correlation are similar for all inputs tested (5ng to 1ug), even though input amounts vary by over three orders of magnitude. These consistent results are recapitulated with the spiked-in ERCC controls at all inputs. Conclusions: Our library preparation method is streamlined and can be used for a wide range of input RNA without any major modifications to the protocol, making it an easy to follow, convenient method for RNA-seq library preparation. In addition, our method has increased sensitivity and specificity, especially for low-abundance transcripts, reduced PCR duplicates and sequence bias, delivering high quality strand-specific data even for low input RNA. Finally, our method is compatible with both poly A-tail enriched and ribosomal RNA depleted samples, and is amenable to large-scale library construction and automation. Citation Format: Keerthana Krishnan, Erbay Yigit, Mehmet Karaca, Deyra Rodriguez, Bradley Langhorst, Timur Shtatland, Daniela Munafo, Pingfang Liu, Lynne Apone, Vaishnavi Panchapakesa, Karen Duggan, Christine Sumner, Christine Rozzi, Fiona Stewart, Laurie Mazzola, Joanna Bybee, Danielle Rivizzigno, Eileen T. Dimalanta, Theodore B. Davis. Low-input transcript profiling with enhanced sensitivity using a highly efficient, low-bias and strand-specific RNA-Seq library preparation method [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 5406. doi:10.1158/1538-7445.AM2017-5406

Sexual identities lie at the heart of social and public health writings about sexual health risk and, as such, are often subject to uncritical assumption and imposition. In this paper, a critique is offered of such discourses along with the equation that sexual identity can predict the nature of practice within and outside the bedroom. In doing so, the authors draw on the findings of two recent independent yet conterminous Australian qualitative studies of sexual identities and practices to examine how young women's and men's identities resist occupying stable, predictable and causal relationships to sexual practices. Finally, the implications of such empirical findings for HIV/AIDS and STI prevention activities are explored.

Academic interest in sexuality and sexual practices has led to a growing body of scholarly work concerned with the deconstruction of heterosexuality. Amid past claims of women's abiding oppression has been more recent work highlighting fluidity and flux in heterosexuality generally, and masculinity and femininity particularly. This article discusses the findings of a recent empirical study of young heterosexual women in the areas of (a) female initiative and desire in heterosexual relations; (b) heterosexual practices; and (c) virginity. It is the consequences of a possible shift in definitions and practices of femininity for young women's experience of heterosex which forms the focus of this discussion.

This article concerns the issues of young women's reputations. By employing a notion of “technology” to investigate “the practices, knowledges and strategies” which surround young women's reputations, this article draws on the recent findings of a 3-year Australian study of young women, heterosexuality, and safe sex practices. By focussing upon the ways in which young women's social lives are both reflected in, and regulated by, a concern for reputation, new insights are generated about the social context in which young people's sexual practice takes place.

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.

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 << 1% (Milbury et al, Clin. Chem. 2012). 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.

Early detection and diagnosis of cancer substantially increases the likelihood for successful treatment. Tools that aid in detecting and diagnosing cancer early, therefore, have the potential to greatly impact the clinical outcome for cancer patients. Next Generation Sequencing (NGS) has emerged as an important tool in this area. The technology is sensitive, fast and high throughput to allow sequencing of many samples at once. Unfortunately, many clinical samples go unanalyzed because they do not yield sufficient quantities of DNA to generate NGS libraries or the libraries generated require so many rounds of PCR amplification that they display extreme sequence bias. Bias not only hampers data analysis, but also increases costs by requiring excess sequencing to obtain sufficient coverage over all relevant genomic regions. To enable the increased use of NGS in the clinic and reduce the amount of sequence bias generated during library preparation, we have developed a PCR free library construction method that uses low quantities of DNA as input. As an initial test of the method, we generated PCR free libraries from 100ng, 50ng and 25ng of human genomic DNA. The libraries where pooled and sequenced on the Illumina NextSeq 500 instrument to approximately 10X coverage. All libraries, irrespective of input amount, showed minimal AT/GC bias and excellent coverage distributions, with most bases covered within 5X of the expected coverage depth. In addition, regions identified as difficult to sequence (Aird, D., et.al., 2011 and Ross, M. G., et.al., 2013) showed coverage at near expected levels for all libraries. This method can easily be adapted for use with extremely low DNA inputs by the introduction of a minimal number of PCR cycles. In fact, we have used this method to construct high quality NGS libraries with picogram quantities of DNA input. Standard library construction methods require DNA inputs of 2ug to 500ng when PCR amplification is omitted. This new method utilizes inputs as low as 25ng to generate high-quality PCR free libraries and picogram quantities when amplification is performed. We are currently investigating the possibility of reducing input levels further and exploring the limits of the method with low quality DNA samples. Interestingly, we have observed substantial sample loss during DNA shearing and reaction cleanup. Samples that do not require fragmentation, such as DNA isolated from plasma (cfDNA) and low quality FFPE DNA, may reduce the input requirements even further. Finally, this new method utilizes low sample and reagent volumes, possibly paving the way for its use in microfluidic devices. Citation Format: Lynne Apone, Pingfang Liu, Vaish Panchapakesa, Deyra Rodriguez, Karen Duggan, Krishnan Keerthana, Nicole Nichols, Yanxia Bei, Julie Menin, Brad Langhorst, Christine Sumner, Christine Chater, Joanna Bybee, Laurie Mazzola, Danielle Rivizzigno, Fiona Stewart, Eileen Dimalanta, Theodore Davis. Enhancing clinical utility of NGS with reduced bias, low DNA input, library construction. [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 3620.

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.

Next Generation Sequencing (NGS) has significantly impacted human genetics, enabling a comprehensive characterization of the human genome as well as a 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. Our objective here was to overcome this challenge by developing NEBNext® Ultra DNA Library Prep Kit, a fast library preparation method. Specifically, we streamlined the workflow utilizing novel NEBNext reagents and adaptors, including a new DNA polymerase that has been optimized to minimize GC bias. As a result of this work, we have developed a simple method for library construction from an amount of DNA as low as 5 ng, which can be used for both intact and fragmented DNA. Moreover, the workflow is compatible with multiple NGS platforms.

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.

Formalin-fixed, paraffin-embedded (FFPE) clinical samples are an invaluable source of information about genetic alterations in human disease, especially cancer. Next generation sequencing is a powerful tool to mine that information. 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. In this study, we investigated the effects of DNA repair on library preparation and sequencing from FFPE samples. We evaluated a repair enzyme mix that is designed to work on a broad range of DNA damage including modified bases, nicks, gaps, and blocked 3′ ends. Results from FFPE DNA samples of varying quality show that DNA repair generally increases library yields by 10-170%, with the largest improvement on samples of poor quality. Careful analysis of sequencing data shows that base calling qualities for all 4 bases are improved upon DNA repair. Aberrant G:C to A:T mutation were significantly reduced, consistent with cytosine deamination being induced during fixation and storage at room temperature. Interestingly, sequencing miscalls for base pair changes not typically associated with fixation were also reduced. We are currently investigating the source of these mutations. With DNA repair a noticeably positive impact on read mapping and read pairing was observed, resulting in the generation of more useable data. Pretreatment of FFPE DNA by the repair enzyme mix is easily implemented upstream of library construction, allowing fast turnaround time and easy automation. We expect that these improvements will enable the analysis of many FFPE samples that would otherwise not be accessible. Citation Format: Pingfang Liu, Lixin Chen, Laurence Ettwiller, Christine Sumner, Fiona Stewart, Eileen Dimalanta, Theodore Davis, Thomas Evans. Repair of challenging FFPE DNA improves library success rate and sequencing quality. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4886. doi:10.1158/1538-7445.AM2015-4886

McrBC, a GTP-requiring, modification-dependent endonuclease of Escherichia coli K-12, specifically recognizes DNA sites of the form 5′ R m C 3′. DNA cleavage normally requires translocation-mediated coordination between two such recognition elements at distinct sites. We have investigated assembly of the cleavage-competent complex with gel-shift and DNase I footprint analysis. In the gel-shift system, McrB L binding resulted in a fast-migrating specific shifted band, in a manner requiring both GTP and Mg 2+ . The binding was specific for methylated DNA and responded to local sequence changes in the same way that cleavage does. Single-stranded DNA competed for McrB L -binding in a modification and sequence-specific fashion. A supershifted species was formed in the presence of McrC and GTPγS. DNase I footprint analysis showed modest cooperativity in binding to two sites, and a two-site substrate displayed protection in non-specific spacer DNA in addition to the recognition elements. The addition of McrC did not affect the footprint obtained. We propose that McrC effects a conformational change in the complex rather than a reorganization of the DNA:protein interface.

A robust Tn7-based in vitro transposition system is described that displays little target site selectivity, allowing the efficient recovery of many different transposon insertions in target DNAs ranging from small plasmids to cosmids to whole genomes. Two miniTn7 derivatives are described that are useful for the analysis of genes: one a derivative for making translational and transcriptional target gene fusions and the other a derivative that can generate 15 bp (5 amino acid) insertions in target DNAs (proteins).

In light of the current and ongoing threats to young women's health of unplanned pregnancy and sexually transmissible diseases (including HIV/AIDS), assured access to appropriate and sensitive health services is of paramount importance. In this article, the results of a recent Victorian study involving rural and urban female secondary school students on issues relating to sexual health are reported. Drawing upon the findings of an extensive self-report questionnaire, young women's attitudes towards and use of health services are presented with a particular focus upon the variations that exist between the rural and urban populations. Apart from issues associated with privacy, which were found to be more crucial to rural young women's decisions to seek medical care, this study found young women's concern about the attitudes of medical and non-medical staff alike, the atmosphere of surgery or clinic, and gender of doctor all to be important factors in their general use of health services, regardless of location.

Next generation sequencing (NGS) has significantly impacted cancer genetics, enabling a comprehensive characterization of genomic abnormalities in the cancer genome at very high resolution. By delivering massive DNA sequences at unprecedented speed and cost, NGS promises to make personalized genome-based diagnosis and targeted cancer therapy 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. This method enables library construction from a minimal quantity of DNA (< 5 ng), and can be used for both intact and fragmented DNA. Moreover, the workflow is compatible with multiple NGS platforms. Citation 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 3180. doi:1538-7445.AM2012-3180

Initial 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. Citation 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