Transposome-based technologies have enabled the streamlined production of sequencer-ready DNA libraries; however, current methods are highly sensitive to the amount and quality of input nucleic acid.
...We describe a new library preparation technology (Nextera DNA Flex) that utilizes a known concentration of transposomes conjugated directly to beads to bind a fixed amount of DNA, and enables direct input of blood and saliva using an integrated extraction protocol. We further report results from libraries generated outside the standard parameters of the workflow, highlighting novel applications for Nextera DNA Flex, including human genome builds and variant calling from below 1 ng DNA input, customization of insert size, and preparation of libraries from short fragments and severely degraded FFPE samples. Using this bead-linked library preparation method, library yield saturation was observed at an input amount of 100 ng. Preparation of libraries from a range of species with varying GC levels demonstrated uniform coverage of small genomes. For large and complex genomes, coverage across the genome, including difficult regions, was improved compared with other library preparation methods. Libraries were successfully generated from amplicons of varying sizes (from 50 bp to 11 kb), however, a decrease in efficiency was observed for amplicons smaller than 250 bp. This library preparation method was also compatible with poor-quality DNA samples, with sequenceable libraries prepared from formalin-fixed paraffin-embedded samples with varying levels of degradation.
In contrast to solution-based library preparation, this bead-based technology produces a normalized, sequencing-ready library for a wide range of DNA input types and amounts, largely obviating the need for DNA quantitation. The robustness of this bead-based library preparation kit and flexibility of input DNA facilitates application across a wide range of fields.
Haplotype-resolved genome sequencing promises to unlock a wealth of information in population and medical genetics. However, for the vast majority of genomes sequenced to date, haplotypes have not ...been determined because of cumbersome haplotyping workflows that require fractions of the genome to be sequenced in a large number of compartments. Here we demonstrate barcode partitioning of long DNA molecules in a single compartment using "on-bead" barcoded tagmentation. The key to the method that we call "contiguity preserving transposition" sequencing on beads (CPTv2-seq) is transposon-mediated transfer of homogenous populations of barcodes from beads to individual long DNA molecules that get fragmented at the same time (tagmentation). These are then processed to sequencing libraries wherein all sequencing reads originating from each long DNA molecule share a common barcode. Single-tube, bulk processing of long DNA molecules with ∼150,000 different barcoded bead types provides a barcode-linked read structure that reveals long-range molecular contiguity. This technology provides a simple, rapid, plate-scalable and automatable route to accurate, haplotype-resolved sequencing, and phasing of structural variants of the genome.
We describe a bead-linked transposome technology used for preparing libraries for WGS (Nextera DNA Flex) as well as targeted enrichment (Nextera Flex for Enrichment). This methodology utilizes a ...known concentration of transposomes conjugated directly to beads to bind a fixed amount of DNA. This therefore offers broad applicability, supporting a wide spectrum of DNA input ranges as well as integrated extraction of blood and saliva samples generating normalized libraries for sequencing to facilitate a quantification-free workflow. Libraries generated outside the standard parameters of the workflow, highlighting novel applications for Nextera DNA Flex, including human genome builds and variant calling from below 1ng DNA input, customization of insert size, and preparation of libraries from short fragments and severely degraded FFPE samples. For large and complex genomes, coverage across the genome, including difficult regions, was improved compared with other library preparation methods. Libraries were successfully generated from amplicons of varying sizes (from 50 bp to 11 kb), however, a decrease in efficiency was observed for amplicons smaller than 250 bp. On-bead tagmentation chemistry is also applicable for targeted sequencing and supports a wide range of DNA input amounts, various sample types, and a broad range of applications, including fixed panels, custom panels, and whole-exome sequencing. Furthermore, NexteraFlex for Enrichmentis compatible with Illumina and third-party enrichment probes/panels, which enables content portability. The innovative NexteraFlex for Enrichment solution combined with the power of Illumina SBS chemistry provides an optimal targeted enrichment and exome sequencing experience. The bead-linked transposomes can therefore be effectively used for WGS as well as for targeted resequencing.
Automated DNA sequencing Wallis, Yvonne; Morrell, Natalie
Methods in molecular biology (Clifton, N.J.),
01/2011, Volume:
688
Journal Article
Fluorescent cycle sequencing of PCR products is a multistage process and several methodologies are available to perform each stage. This chapter will describe the more commonly utilised ...dye-terminator cycle sequencing approach using BigDye® terminator chemistry (Applied Biosystems) ready for analysis on a 3730 DNA genetic analyzer. Even though DNA sequencing is one of the most common and robust techniques performed in molecular laboratories it may not always produce desirable results. The causes of the most common problems will also be discussed in this chapter.
Currently, no safe and effective pharmacologic interventions exist for acute kidney injury (AKI). One reason may be that heterogeneity exists within the AKI population, thereby hampering the ...identification of specific pathophysiologic pathways and therapeutic targets.
The aim of this study was to identify and test whether AKI subphenotypes have prognostic and therapeutic implications.
First, latent class analysis methodology was applied independently in two critically ill populations (discovery n = 794 and replication n = 425) with AKI. Second, a parsimonious classification model was developed to identify AKI subphenotypes. Third, the classification model was applied to patients with AKI in VASST (Vasopressin and Septic Shock Trial; n = 271), and differences in treatment response were determined. In all three populations, AKI was defined using serum creatinine and urine output.
A two-subphenotype latent class analysis model had the best fit in both the discovery (P = 0.004) and replication (P = 0.004) AKI groups. The risk of 7-day renal nonrecovery and 28-day mortality was greater with AKI subphenotype 2 (AKI-SP2) relative to AKI subphenotype 1 (AKI-SP1). The AKI subphenotypes discriminated risk for poor clinical outcomes better than the Kidney Disease: Improving Global Outcomes stages of AKI. A three-variable model that included markers of endothelial dysfunction and inflammation accurately determined subphenotype membership (C-statistic 0.92). In VASST, vasopressin compared with norepinephrine was associated with improved 90-day mortality in AKI-SP1 (27% vs. 46%, respectively; P = 0.02), but no significant difference was observed in AKI-SP2 (45% vs. 49%, respectively; P = 0.99) and the P value for interaction was 0.05.
This analysis identified two molecularly distinct AKI subphenotypes with different clinical outcomes and responses to vasopressin therapy. Identification of AKI subphenotypes could improve risk prognostication and may be useful for predictive enrichment in clinical trials.
Inflammation is a feature of pulmonary arterial hypertension (PAH), and increased circulating levels of cytokines are reported in patients with PAH. However, to date, no information exists on the ...significance of elevated cytokines or their potential as biomarkers. We sought to determine the levels of a range of cytokines in PAH and to examine their impact on survival and relationship to hemodynamic indexes.
We measured levels of serum cytokines (tumor necrosis factor-alpha, interferon-gamma and interleukin-1beta, -2, -4, -5, -6, -8, -10, -12p70, and -13) using ELISAs in idiopathic and heritable PAH patients (n=60). Concurrent clinical data included hemodynamics, 6-minute walk distance, and survival time from sampling to death or transplantation. Healthy volunteers served as control subjects (n=21). PAH patients had significantly higher levels of interleukin-1beta, -2, -4, -6, -8, -10, and -12p70 and tumor necrosis factor-alpha compared with healthy control subjects. Kaplan-Meier analysis showed that levels of interleukin-6, 8, 10, and 12p70 predicted survival in patients. For example, 5-year survival with interleukin-6 levels of >9 pg/mL was 30% compared with 63% for patients with levels < or = 9 pg/mL (P=0.008). In this PAH cohort, cytokine levels were superior to traditional markers of prognosis such as 6-minute walk distance and hemodynamics.
This study illustrates dysregulation of a broad range of inflammatory mediators in idiopathic and familial PAH and demonstrates that cytokine levels have a previously unrecognized impact on patient survival. They may prove to be useful biomarkers and provide insight into the contribution of inflammation in PAH.
Background: There are currently no licensed medical therapies for inoperable chronic thromboembolic pulmonary hypertension (CTEPH).
Methods: In this double-blind, placebo-controlled pilot study, 19 ...subjects with inoperable CTEPH were randomly assigned to sildenafil
or placebo for 12 weeks. The primary end point was change in 6-min walking distance (6MWD). Secondary end points included
changes in World Health Organization (WHO) class, cardiopulmonary hemodynamics, quality of life (QOL) scores, and N-terminal
pro brain natriuretic peptide (NT-proBNP). All subjects were transferred to open-label sildenafil at the end of the study
and offered repeat assessment at 12 months.
Results: There were no significant differences between the two groups with respect to change in exercise capacity. However significant
improvements were seen in WHO class and pulmonary vascular resistance (PVR). Seventeen subjects were eligible for reassessment
at 12 months and demonstrated significant improvements in 6MWD, activity and symptom components of QOL, cardiac index, PVR,
and NT-proBNP.
Conclusions: Although this pilot study was insufficiently powered to test the primary end point, it did suggest beneficial effects in
favor of sildenafil in several secondary end points at both 3 months and 12 months. Further larger-scale trials of sildenafil
in inoperable CTEPH are required to confirm these findings and potentially increase the treatment options available for this
devastating disease.
Trial registration: The study protocol was registered with the UK National Research Register database (publication ID N0542136603).
inoperable
pulmonary endarterectomy
pulmonary hypertension
sildenafil
thromboembolism
Background The research criteria for subjective cognitive decline (SCD) exclude mild cognitive impairment (MCI), but do not stipulate the use of specific MCI criteria. This study compared different ...approaches to defining (i.e., excluding) MCI during the ascertainment of SCD, focusing on the impact on dementia incidence rates in SCD. Methods This cohort study utilized routine healthcare data collected in the Essex Memory Clinic from 1999 to 2023. Two different operationalizations of the SCD criteria were used to categorize the cohort into two SCD patient samples. One sample was based on local clinical practice - MCI was excluded according to the Winblad criteria (this sample was termed SCD.sub.Winblad). The other sample was created via the retrospective application of the Jak/Bondi criteria for the exclusion of MCI (termed SCD.sub.Jak/Bondi). Only patients aged greater than or equal to 55 years at baseline with greater than or equal to 12 months follow-up were considered for inclusion. The initial clinical/demographic characteristics of the samples were compared. Rates of incident dementia were calculated for each sample, and unadjusted and Mantel-Haenszel-adjusted incidence rate ratios were calculated to compare dementia incidence between the SCD samples. Results The Essex Memory Clinic database included 2,233 patients in total. The SCD and study eligibility criteria were used to select SCD.sub.Winblad (n = 86) and SCD.sub.Jak/Bondi (n = 185) samples from the database. Median follow-up (3 years) did not differ between the two samples. The SCD.sub.Jak/Bondi sample was significantly older than the SCD.sub.Winblad at first assessment (median age: 74 versus 70 years) and had poorer scores on tests of global cognition, immediate and delayed verbal recall, and category fluency. Following adjustment for age, the dementia incidence rate ratio 95% confidence interval was 3.7 1.5 to 9.3, indicating a significantly greater rate of progression to dementia in SCD.sub.Jak/Bondi. Conclusions This study highlights that the approach used to ascertain SCD has important implications for both SCD phenotypes and prognosis. This underscores the importance of how MCI is operationalized within SCD studies. More broadly, the findings add to a growing body of work indicating that objective cognition should not be overlooked in SCD, and offer a potential explanation for the heterogeneity across the SCD prognostic literature. Keywords: Mild cognitive impairment, Subjective cognitive decline, Dementia, Incidence, Cognitive testing, Criteria
No direct comparisons of clinical features, laboratory values, and outcomes between critically ill patients with coronavirus disease (COVID-19) and patients with influenza in the United States have ...been reported.
To evaluate the risk of mortality comparing critically ill patients with COVID-19 with patients with seasonal influenza.
We retrospectively identified patients admitted to the intensive care units (ICUs) at two academic medical centers with laboratory-confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or influenza A or B infections between January 1, 2019, and April 15, 2020. The clinical data were obtained by medical record review. All patients except one had follow-up to hospital discharge or death. We used relative risk regression adjusting for age, sex, number of comorbidities, and maximum sequential organ failure scores on Day 1 in the ICU to determine the risk of hospital mortality and organ dysfunction in patients with COVID-19 compared with patients with influenza.
We identified 65 critically ill patients with COVID-19 and 74 patients with influenza. The mean (±standard deviation) age in each group was 60.4 ± 15.7 and 56.8 ± 17.6 years, respectively. Patients with COVID-19 were more likely to be male, have a higher body mass index, and have higher rates of chronic kidney disease and diabetes. Of the patients with COVID-19, 37% identified as Hispanic, whereas 10% of the patients with influenza identified as Hispanic. A similar proportion of patients had fevers (∼40%) and lymphopenia (∼80%) on hospital presentation. The rates of acute kidney injury and shock requiring vasopressors were similar between the groups. Although the need for invasive mechanical ventilation was also similar in both groups, patients with COVID-19 had slower improvements in oxygenation, longer durations of mechanical ventilation, and lower rates of extubation than patients with influenza. The hospital mortality was 40% in patients with COVID-19 and 19% in patients with influenza (adjusted relative risk, 2.13; 95% confidence interval, 1.24-3.63;
= 0.006).
The need for invasive mechanical ventilation was common in patients in the ICU for COVID-19 and influenza. Compared with those with influenza, patients in the ICU with COVID-19 had worse respiratory outcomes, including longer duration of mechanical ventilation. In addition, patients with COVID-19 were at greater risk for in-hospital mortality, independent of age, sex, comorbidities, and ICU severity of illness.
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