ABSTRACT
Effective vaccines induce high-affinity memory B cells and durable antibody responses through accelerated mechanisms of natural selection. Secondary changes in antibody repertoires after vaccine boosts suggest progressive rediversification of B cell receptors (BCRs), but the underlying mechanisms remain unresolved. Here, the integrated specificity and function of individual memory B cell progeny revealed ongoing evolution of polyclonal antibody specificities through germinal center (GC)-specific transcriptional activity. At the clonal and subclonal levels, single-cell expression of the genes encoding the costimulatory molecule CD83 and the DNA polymerase Polη segregated the secondary GC transcriptional program into four stages that regulated divergent mechanisms of memory BCR evolution. Our studies demonstrate that vaccine boosts reactivate a cyclic program of GC function in class-switched memory B cells to remodel existing antibody specificities and enhance durable immunological protection.
Subject(s)
B-Lymphocytes/immunology , Germinal Center/immunology , Immunologic Memory/immunology , Receptors, Antigen, B-Cell/immunology , Animals , Antibodies/immunology , Antibody Formation/immunology , Antigens, CD/immunology , DNA-Directed DNA Polymerase/immunology , Immunoglobulin Class Switching/immunology , Immunoglobulins/immunology , Membrane Glycoproteins/immunology , Mice , Mice, Inbred C57BL , Transcription, Genetic/immunology , CD83 AntigenABSTRACT
Antibodies are produced across multiple isotypes with distinct properties that coordinate initial antigen clearance and confer long-term antigen-specific immune protection. Here, we interrogate the molecular programs of isotype-specific murine plasma cells (PC) following helper T cell-dependent immunization and within established steady-state immunity. We developed a single-cell-indexed and targeted molecular strategy to dissect conserved and divergent components of the rapid effector phase of antigen-specific IgM+ versus inflammation-modulating programs dictated by type 1 IgG2a/b+ PC differentiation. During antibody affinity maturation, the germinal center (GC) cycle imparts separable programs for post-GC type 2 inhibitory IgG1+ and type 1 inflammatory IgG2a/b+ PC to direct long-term cellular function. In the steady state, two subsets of IgM+ and separate IgG2b+ PC programs clearly segregate from splenic type 3 IgA+ PC programs that emphasize mucosal barrier protection. These diverse isotype-specific molecular pathways of PC differentiation control complementary modules of antigen clearance and immune protection that could be selectively targeted for immunotherapeutic applications and vaccine design.
Subject(s)
Cell Differentiation , Germinal Center , Plasma Cells , Animals , Antigens , Immunoglobulin G/genetics , Immunoglobulin M , Mice , Plasma Cells/cytology , Single-Cell Analysis , T-Lymphocytes, Helper-InducerABSTRACT
Bisulfite sequencing detects 5mC and 5hmC at single-base resolution. However, bisulfite treatment damages DNA, which results in fragmentation, DNA loss, and biased sequencing data. To overcome these problems, enzymatic methyl-seq (EM-seq) was developed. This method detects 5mC and 5hmC using two sets of enzymatic reactions. In the first reaction, TET2 and T4-BGT convert 5mC and 5hmC into products that cannot be deaminated by APOBEC3A. In the second reaction, APOBEC3A deaminates unmodified cytosines by converting them to uracils. Therefore, these three enzymes enable the identification of 5mC and 5hmC. EM-seq libraries were compared with bisulfite-converted DNA, and each library type was ligated to Illumina adaptors before conversion. Libraries were made using NA12878 genomic DNA, cell-free DNA, and FFPE DNA over a range of DNA inputs. The 5mC and 5hmC detected in EM-seq libraries were similar to those of bisulfite libraries. However, libraries made using EM-seq outperformed bisulfite-converted libraries in all specific measures examined (coverage, duplication, sensitivity, etc.). EM-seq libraries displayed even GC distribution, better correlations across DNA inputs, increased numbers of CpGs within genomic features, and accuracy of cytosine methylation calls. EM-seq was effective using as little as 100 pg of DNA, and these libraries maintained the described advantages over bisulfite sequencing. EM-seq library construction, using challenging samples and lower DNA inputs, opens new avenues for research and clinical applications.
ABSTRACT
Antibody class defines function in B cell immunity, but how class is propagated into B cell memory remains poorly understood. Here we demonstrate that memory B cell subsets unexpectedly diverged across antibody class through differences in the effects of major transcriptional regulators. Conditional genetic deletion of the gene encoding the transcription factor T-bet selectively blocked the formation and antigen-specific response of memory B cells expressing immunoglobulin G2a (IgG2a) in vivo. Cell-intrinsic expression of T-bet regulated expression of the transcription factor STAT1, steady-state cell survival and transcription of IgG2a-containing B cell antigen receptors (BCRs). In contrast, the transcription factor RORα and not T-bet was expressed in IgA(+) memory B cells, with evidence that knockdown of RORα mRNA expression and chemical inhibition of transcriptional activity also resulted in lower survival and BCR expression of IgA(+) memory B cells. Thus, divergent transcriptional regulators dynamically maintain subset integrity to promote specialized immune function in class-specific memory B cells.
Subject(s)
B-Lymphocyte Subsets/immunology , B-Lymphocytes/immunology , Immunoglobulin Class Switching/immunology , Immunologic Memory/immunology , Receptor Tyrosine Kinase-like Orphan Receptors/immunology , T-Box Domain Proteins/immunology , Animals , B-Lymphocytes/classification , Flow Cytometry , Immunoglobulin A/immunology , Immunoglobulin G/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , RNA, Messenger/chemistry , RNA, Messenger/genetics , Receptor Tyrosine Kinase-like Orphan Receptors/genetics , Receptors, Antigen, B-Cell/immunology , Reverse Transcriptase Polymerase Chain Reaction , STAT1 Transcription Factor/immunology , Specific Pathogen-Free Organisms , T-Box Domain Proteins/genetics , Transcription, Genetic/immunologyABSTRACT
BACKGROUND: Anxiety in pregnancy and after giving birth (the perinatal period) is highly prevalent but under-recognised. Robust methods of assessing perinatal anxiety are essential for services to identify and treat women appropriately. AIMS: To determine which assessment measures are most psychometrically robust and effective at identifying women with perinatal anxiety (primary objective) and depression (secondary objective). METHOD: We conducted a prospective longitudinal cohort study of 2243 women who completed five measures of anxiety and depression (Generalized Anxiety Disorder scale (GAD) two- and seven-item versions; Whooley questions; Clinical Outcomes in Routine Evaluation (CORE-10); and Stirling Antenatal Anxiety Scale (SAAS)) during pregnancy (15 weeks, 22 weeks and 31 weeks) and after birth (6 weeks). To assess diagnostic accuracy a sample of 403 participants completed modules of the Mini-International Neuropsychiatric Interview (MINI). RESULTS: The best diagnostic accuracy for anxiety was shown by the CORE-10 and SAAS. The best diagnostic accuracy for depression was shown by the CORE-10, SAAS and Whooley questions, although the SAAS had lower specificity. The same cut-off scores for each measure were optimal for identifying anxiety or depression (SAAS ≥9; CORE-10 ≥9; Whooley ≥1). All measures were psychometrically robust, with good internal consistency, convergent validity and unidimensional factor structure. CONCLUSIONS: This study identified robust and effective methods of assessing perinatal anxiety and depression. We recommend using the CORE-10 or SAAS to assess perinatal anxiety and the CORE-10 or Whooley questions to assess depression. The GAD-2 and GAD-7 did not perform as well as other measures and optimal cut-offs were lower than currently recommended.
Subject(s)
Anxiety Disorders , Anxiety , Female , Pregnancy , Humans , Prospective Studies , Longitudinal Studies , Anxiety Disorders/diagnosis , Anxiety Disorders/psychology , Anxiety/diagnosis , Psychiatric Status Rating Scales , PsychometricsABSTRACT
BACKGROUND: Anxiety in pregnancy and postpartum is highly prevalent but under-recognised. To identify perinatal anxiety, assessment tools must be acceptable, relevant, and easy to use for women in the perinatal period. METHODS: To determine the acceptability and ease of use of anxiety measures to pregnant or postpartum women (n = 41) we examined five versions of four measures: the Generalised Anxiety Disorder scale (GAD) 2-item and 7-item versions; Whooley questions; Clinical Outcomes in Routine Evaluation (CORE-10); and Stirling Antenatal Anxiety Scale (SAAS). Cognitive interviews were used to examine ease of comprehension, judgement, retrieval and responding. RESULTS: All measures were acceptable. Some items were deemed less relevant to the perinatal period e.g., difficulties sleeping. Ease of comprehension, judgement, retrieval and responding varied, with all measures having strengths and weaknesses. The SAAS and CORE-10 had the lowest mean number of problematic components. The GAD had the highest mean number of problematic componentsâ. Non-binary response options were preferred. Preferences for time frames (e.g. one week, one month) varied. Qualitative data provides in-depth information on responses to each measure. CONCLUSIONS: Findings can be used to inform clinical guidelines and research on acceptable anxiety assessment in pregnancy and after birth.
Subject(s)
Anxiety , Pregnancy Complications , Humans , Female , Pregnancy , Adult , Pregnancy Complications/psychology , Pregnancy Complications/diagnosis , Anxiety/psychology , Anxiety/diagnosis , Postpartum Period/psychology , Psychiatric Status Rating Scales/standards , Anxiety Disorders/diagnosis , Anxiety Disorders/psychology , Young Adult , Interviews as TopicABSTRACT
B lymphocytes differentiate into antibody-secreting cells under the antigen-specific control of follicular helper T cells (T(FH) cells). Here we demonstrate that isotype-switched plasma cells expressed major histocompatibility complex (MHC) class II, the costimulatory molecules CD80 and CD86, and the intracellular machinery required for antigen presentation. Antigen-specific plasma cells accessed, processed and presented sufficient antigen in vivo to induce multiple helper T cell functions. Notably, antigen-primed plasma cells failed to induce interleukin 21 (IL-21) or the transcriptional repressor Bcl-6 in naive helper T cells and actively decreased these key molecules in antigen-activated T(FH) cells. Mice lacking plasma cells showed altered T(FH) cell activity, which provided evidence of this negative feedback loop. Hence, antigen presentation by plasma cells defines a previously unknown layer of cognate regulation that limits the antigen-specific T(FH) cell program that controls ongoing B cell immunity.
Subject(s)
Antigen Presentation/immunology , Lymphocyte Activation/immunology , Plasma Cells/immunology , T-Lymphocytes, Helper-Inducer/immunology , Adaptive Immunity , Animals , Cell Separation , Enzyme-Linked Immunospot Assay , Flow Cytometry , Immunologic Memory , Mice , Mice, Inbred C57BL , Microscopy, Confocal , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
OBJECTIVE: Induction of puberty with exogenous oestrogen results in considerable variability in final uterine and breast volumes. We set out to quantify the variability of these two outcome measures with a view to establishing monitoring methods that could be used to individualise treatment protocols. DESIGN: A prospective observational study. PARTICIPANTS: Sixteen participants with pubertal delay and primary amenorrhoea, due to hypogonadism were recruited from paediatric gynaecology and endocrinology clinics at University College London Hospital. A standardised protocol of transdermal 17ß oestradiol (17ßE) was used (Evorel™), with a starting dose of 12.5 mcg increasing to 25 mcg (patch changed twice weekly) after 4 months. Follow up was every 2 months for a total of 8 months. MEASUREMENTS: Uterine dimensions using ultrasound, oestradiol concentrations and breast development assessed by both Tanner staging and 3D photographic imaging. RESULTS: After 8 months of treatment, the changes in oestradiol concentrations (0-174 pmol), uterine volume growth (4.4-16.4 ml) and breast volume (1.76-140.1 ml) varied greatly between individuals. Of uterine parameters, transverse uterine diameter was most closely associated with serum oestradiol levels at 8 months (beta standardised coefficient = 0.80, p = .001). Change in breast volume was associated with age of treatment initiation (beta standardised coefficient 0.55 p = .04). CONCLUSIONS: We demonstrate variation in response to exogenous oestrogen, emphasising the necessity for individualised dose titration. In the absence of sensitive oestradiol assays, uterine transverse measurements may be used as a surrogate marker of oestrogen sensitivity to guide early dose adjustment. 3D breast imaging may provide a quantitative assessment of breast development to complement Tanner breast staging.
Subject(s)
Puberty, Precocious , Uterus , Child , Estradiol , Estrogens , Female , Humans , Puberty/physiology , Uterus/diagnostic imagingABSTRACT
Helper T cell induced plasma cells (PCs) that secrete class-switched neutralizing antibody are paramount to effective immunity. Following class-switch recombination (CSR), antigen-activated B cells differentiate into extrafollicular PCs or mature in germinal centers (GCs) to produce high-affinity memory B cells and follicular PCs. Many studies focus on the core transcriptional programs that drive central PC functions of longevity and antibody secretion. However, it is becoming clear that these central programs are further subdivided across antibody isotype with separable transcriptional trajectories. Divergent functions emerge at CSR, persist through PC terminal differentiation and further assort memory PC function following antigen recall. Here, we emphasize recent work that assorts divergent isotype-specific PC function across four major modules of immune protection.
Subject(s)
Plasma Cells/immunology , T-Lymphocytes, Helper-Inducer/immunology , Animals , HumansABSTRACT
How follicular helper T cells (T(FH) cells) differentiate to regulate B cell immunity is critical for effective protein vaccination. Here we define three transcription factor T-bet-expressing antigen-specific effector helper T cell subsets with distinguishable function, migratory properties and developmental programming in vivo. Expression of the transcriptional repressor Blimp-1 distinguished T zone 'lymphoid' effector helper T cells (CD62L(hi)CCR7(hi)) from CXCR5(lo) 'emigrant' effector helper T cells and CXCR5(hi) 'resident' T(FH) cells expressing the transcriptional repressor Bcl-6 (CD62L(lo)CCR7(lo)). We then show by adoptive transfer and intact polyclonal responses that helper T cells with the highest specific binding of peptide-major histocompatibility complex class II and the most restricted T cell antigen receptor junctional diversity 'preferentially' developed into the antigen-specific effector T(FH) compartment. Our studies demonstrate a central function for differences in the binding strength of the T cell antigen receptor in the antigen-specific mechanisms that 'program' specialized effector T(FH) function in vivo.
Subject(s)
Receptors, Antigen, T-Cell/immunology , T-Lymphocytes, Helper-Inducer/immunology , Adjuvants, Immunologic/pharmacology , Adoptive Transfer , Animals , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Cell Differentiation/immunology , Cytokines/biosynthesis , DNA-Binding Proteins/immunology , DNA-Binding Proteins/metabolism , Histocompatibility Antigens Class II/metabolism , L-Selectin/immunology , Lymph Nodes/immunology , Mice , Mice, Transgenic , Positive Regulatory Domain I-Binding Factor 1 , Proto-Oncogene Proteins c-bcl-6 , Receptors, Antigen, T-Cell/metabolism , T-Box Domain Proteins/metabolism , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , T-Lymphocytes, Helper-Inducer/metabolism , T-Lymphocytes, Helper-Inducer/transplantation , Transcription Factors/biosynthesis , Transcription Factors/immunologyABSTRACT
Anatomical information and pathologies have been conveyed through the medium of medical illustrations for centuries. In the formative years of British neurosurgery, Professor Norman Dott (1897-1973) utilised medical illustrations as a means of documenting neurosurgical advances and conveying pathological-anatomical correlation. He commissioned a vast number of medical illustrations over the course of his career, ultimately producing a diverse collection of items, most of which is cared for by Lothian Health Services Archive (LHSA), Edinburgh, Scotland. In this study, the original material from Dott's personal collection was audited. Of 172 stand-alone drawings, 84 were categorised and analysed. The findings are a reflection of Dott's expertise as an academic and a surgeon. Spanning the years 1925-1968, a wide range of pathologies and procedures are depicted including intracranial aneurysms and their ligation, an area in which Dott was renowned for pioneering surgical advances. The collection stands as a testament to Dott's emphasis upon medical illustration to communicate the intricacies and complexities of his field, providing valuable insight into clinical and surgical practice in neurosurgery when the specialty was in its juvenescence. In order to illuminate the connections between biography and specialism that generated an extraordinary visual archive, this study considers the early life and work of Norman Dott and the influence of Harvey Cushing on Dott's prioritisation of visual documentation of surgical practice. It explores the impact of German-American medical artist Max Brödel on the UK, and especially on the artists employed by Dott, before presenting a short review of the medical illustrations they created.
Subject(s)
Intracranial Aneurysm , Neurosurgery , Humans , Male , Medical Illustration , Neurosurgical Procedures , ScotlandABSTRACT
Recent studies have highlighted the promise of targeting tumor neoantigens to generate potent antitumor immune responses and provide strong motivation for improving our understanding of antigen-T-cell receptor (TCR) interactions. Advances in single-cell sequencing technologies have opened the door for detailed investigation of the TCR repertoire, providing paired information from TCRα and TCRß, which together determine specificity. However, a need remains for efficient methods to assess the specificity of discovered TCRs. We developed a streamlined approach for matching TCR sequences with cognate antigen through on-demand cloning and expression of TCRs and screening against candidate antigens. Here, we first demonstrate the system's capacity to identify viral-antigen-specific TCRs and compare the functional avidity of TCRs specific for a given antigen target. We then apply this system to identify neoantigen-specific TCR sequences from patients with melanoma treated with personalized neoantigen vaccines and characterize functional avidity of neoantigen-specific TCRs. Furthermore, we use a neoantigen-prediction pipeline to show that an insertion-deletion mutation in a putative chronic lymphocytic leukemia (CLL) driver gives rise to an immunogenic neoantigen mut-MGA, and use this approach to identify the mut-MGA-specific TCR sequence. This approach provides a means to identify and express TCRs, and then rapidly assess antigen specificity and functional avidity of a reconstructed TCR, which can be applied for monitoring antigen-specific T-cell responses, and potentially for guiding the design of effective T-cell-based immunotherapies.
Subject(s)
Antigens, Neoplasm/immunology , Receptors, Antigen, T-Cell/immunology , T-Cell Antigen Receptor Specificity , Cancer Vaccines/therapeutic use , Cells, Cultured , Cloning, Molecular/methods , HEK293 Cells , Humans , Jurkat Cells , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Melanoma/immunology , Melanoma/therapy , Receptors, Antigen, T-Cell/geneticsABSTRACT
Information is now available in real time from a multitude of sources. Twitter provides one effective means to broadcast images with short captions instantly and everywhere. Last year we began using Twitter to convey our excitement with the biological sciences, and discovered a new means to contribute, connect, and conference with a broader global scientific community and beyond. Here we share this experience, and invite you to join in the conversation.
Subject(s)
Information Dissemination , Science/education , Social Media , Animals , Audiovisual Aids , Communication , Communications Media , HumansABSTRACT
Follicular helper T (Tfh) cells are the class of effector T helper cells that regulates the step-wise development of antigen-specific B cell immunity in vivo. Deployment of CXCR5+ Tfh cells to B cell zones of lymphoid tissues and stable cognate interactions with B cells are central to the delivery of antigen-specific Tfh cell function. Here, we review recent advances that have helped to unravel distinctive elements of developmental programming for Tfh cells and unique effector Tfh cell functions focused on antigen-primed B cells. Understanding the regulatory functions of Tfh cells in the germinal center and the subsequent regulation of memory B cell responses to antigen recall represent the frontiers of this research area with the potential to alter fundamentally the design of future vaccines.
Subject(s)
Lymphocyte Subsets/cytology , T-Lymphocytes, Helper-Inducer/cytology , Animals , B-Lymphocytes/immunology , Cell Differentiation , Cell Lineage , Humans , Immunity , Lymphocyte Subsets/immunology , Receptors, CXCR5/metabolism , T-Lymphocytes, Helper-Inducer/immunologyABSTRACT
How T cell receptor (TCR) specificity evolves in vivo after protein vaccination is central to the development of helper T (Th) cell function. Most models of clonal selection in the Th cell compartment favor TCR affinity-based thresholds. Here, we demonstrated that depot-forming vaccine adjuvants did not require Toll-like receptor (TLR) agonists to induce clonal dominance in antigen-specific Th cell responses. However, readily dispersible adjuvants using TLR-9 and TLR-4 agonists skewed TCR repertoire usage by increasing TCR selection thresholds and enhancing antigen-specific clonal expansion. In this manner, vaccine adjuvants control the local accumulation of Th cells expressing TCR with the highest peptide MHC class II binding. Clonal composition was altered by mechanisms that blocked the local propagation of clonotypes independently of antigen dose and not as a consequence of interclonal competition. This capacity of adjuvants to modify antigen-specific Th cell clonal composition has fundamental implications for the design of future protein subunit vaccines.
Subject(s)
Adjuvants, Immunologic , Cytochromes c/metabolism , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes, Helper-Inducer/immunology , Toll-Like Receptors/metabolism , Vaccines/immunology , Animals , Cytochromes c/immunology , Lymphocyte Activation , Mice , Mice, Congenic , Mice, Transgenic , Receptors, Antigen, T-Cell/immunology , T-Lymphocytes, Helper-Inducer/metabolism , Toll-Like Receptors/agonists , Toll-Like Receptors/immunology , Vaccines, Subunit/immunologySubject(s)
Hysteria/history , Nervous System Diseases/history , Neurology/history , History, 20th Century , HumansABSTRACT
This study examined whether beliefs about face recognition ability differentially influence memory retrieval in older compared to young adults. Participants evaluated their ability to recognise faces and were also given information about their ability to perceive and recognise faces. The information was ostensibly based on an objective measure of their ability, but in actuality, participants had been randomly assigned the information they received (high ability, low ability or no information control). Following this information, face recognition accuracy for a set of previously studied faces was measured using a remember-know memory paradigm. Older adults rated their ability to recognise faces as poorer compared to young adults. Additionally, negative information about face recognition ability improved only older adults' ability to recognise a previously seen face. Older adults were also found to engage in more familiarity than item-specific processing than young adults, but information about their face recognition ability did not affect face processing style. The role that older adults' memory beliefs have in the meta-cognitive strategies they employ is discussed.
Subject(s)
Aging/psychology , Mental Recall/physiology , Metacognition/physiology , Pattern Recognition, Visual/physiology , Recognition, Psychology/physiology , Adult , Age Factors , Aged , Face , Female , Humans , Male , Middle Aged , Photic Stimulation , Young AdultABSTRACT
Eliminating the bacterial cloning step has been a major factor in the vastly improved efficiency of massively parallel sequencing approaches. However, this also has made it a technical challenge to produce the modern equivalent of the Fosmid- or BAC-end sequences that were crucial for assembling and analyzing complex genomes during the Sanger-based sequencing era. To close this technology gap, we developed Fosill, a method for converting Fosmids to Illumina-compatible jumping libraries. We constructed Fosmid libraries in vectors with Illumina primer sequences and specific nicking sites flanking the cloning site. Our family of pFosill vectors allows multiplex Fosmid cloning of end-tagged genomic fragments without physical size selection and is compatible with standard and multiplex paired-end Illumina sequencing. To excise the bulk of each cloned insert, we introduced two nicks in the vector, translated them into the inserts, and cleaved them. Recircularization of the vector via coligation of insert termini followed by inverse PCR generates a jumping library for paired-end sequencing with 101-base reads. The yield of unique Fosmid-sized jumps is sufficiently high, and the background of short, incorrectly spaced and chimeric artifacts sufficiently low, to enable applications such as mapping of structural variation and scaffolding of de novo assemblies. We demonstrate the power of Fosill to map genome rearrangements in a cancer cell line and identified three fusion genes that were corroborated by RNA-seq data. Our Fosill-powered assembly of the mouse genome has an N50 scaffold length of 17.0 Mb, rivaling the connectivity (16.9 Mb) of the Sanger-sequencing based draft assembly.
Subject(s)
Escherichia coli/genetics , Genetic Vectors/genetics , Genome, Bacterial , Genome, Fungal , Genomic Library , Schizosaccharomyces/genetics , Sequence Analysis, DNA/methods , Animals , Gene Rearrangement , Mice , Mice, Inbred C57BLABSTRACT
Massively parallel DNA sequencing technologies are revolutionizing genomics by making it possible to generate billions of relatively short (~100-base) sequence reads at very low cost. Whereas such data can be readily used for a wide range of biomedical applications, it has proven difficult to use them to generate high-quality de novo genome assemblies of large, repeat-rich vertebrate genomes. To date, the genome assemblies generated from such data have fallen far short of those obtained with the older (but much more expensive) capillary-based sequencing approach. Here, we report the development of an algorithm for genome assembly, ALLPATHS-LG, and its application to massively parallel DNA sequence data from the human and mouse genomes, generated on the Illumina platform. The resulting draft genome assemblies have good accuracy, short-range contiguity, long-range connectivity, and coverage of the genome. In particular, the base accuracy is high (≥99.95%) and the scaffold sizes (N50 size = 11.5 Mb for human and 7.2 Mb for mouse) approach those obtained with capillary-based sequencing. The combination of improved sequencing technology and improved computational methods should now make it possible to increase dramatically the de novo sequencing of large genomes. The ALLPATHS-LG program is available at http://www.broadinstitute.org/science/programs/genome-biology/crd.
Subject(s)
Algorithms , Genomics/methods , Sequence Analysis, DNA/methods , Software , Animals , Genome/genetics , Humans , Internet , Mice , Reproducibility of ResultsABSTRACT
BACKGROUND: Anxiety in pregnancy and postnatally is highly prevalent but under-recognized. To identify perinatal anxiety, assessment tools must be acceptable to women who are pregnant or postnatal. METHODS: A qualitative study of women's experiences of anxiety and mental health assessment during pregnancy and after birth and views on the acceptability of perinatal anxiety assessment. Semi-structured interviews were conducted with 41 pregnant or postnatal women. Results were analysed using Sekhon et al.'s acceptability framework, as well as inductive coding of new or emergent themes. RESULTS: Women's perceptions of routine assessment for perinatal anxiety were generally favourable. Most participants thought assessment was needed and that the benefits outweighed potential negative impacts, such as unnecessary referrals to specialist services. Six themes were identified of: (1) Raising awareness; (2) Improving support; (3) Surveillance and stigma; (4) Gatekeeping; (5) Personalized care and (6) Trust. Assessment was seen as a tool for raising awareness about mental health during the perinatal period and a mechanism for normalizing discussions about mental health more generally. However, views on questionnaire assessments themselves were mixed, with some participants feeling they could become an administrative 'tick box' exercise that depersonalizes care and does not provide a space to discuss mental health problems. CONCLUSION: Routine assessment of perinatal anxiety was generally viewed as positive and acceptable; however, this was qualified by the extent to which it was informed and personalized as a process. Approaches to assessment should ideally be flexible, tailored across the perinatal period and embedded in continuity of care.