We present a hierarchical genome-assembly process (HGAP) for high-quality de novo microbial genome assemblies using only a single, long-insert shotgun DNA library in conjunction with Single Molecule, ...Real-Time (SMRT) DNA sequencing. Our method uses the longest reads as seeds to recruit all other reads for construction of highly accurate preassembled reads through a directed acyclic graph-based consensus procedure, which we follow with assembly using off-the-shelf long-read assemblers. In contrast to hybrid approaches, HGAP does not require highly accurate raw reads for error correction. We demonstrate efficient genome assembly for several microorganisms using as few as three SMRT Cell zero-mode waveguide arrays of sequencing and for BACs using just one SMRT Cell. Long repeat regions can be successfully resolved with this workflow. We also describe a consensus algorithm that incorporates SMRT sequencing primary quality values to produce de novo genome sequence exceeding 99.999% accuracy.
Background
The aim of this study was to evaluate outcome and safety of free flaps to the upper extremity by pooled, summarized rates, compared with the existing pooled results of the lower extremity ...from literature, and to improve the level of evidence in a meta‐analysis.
Methods
A literature search of PubMed and Embase was conducted addressing publications between January 2000 and April 2018. Publications were selected by inclusion/exclusion criteria on safety and outcome. Postoperative complications were statistically analyzed: flap loss, vessel thrombosis; recipient‐site infection, hematoma, seroma, wound dehiscence, dysesthesia; donor‐site morbidity.
Results
Two hundred and seventy‐nine patients with 283 free flaps from 23 publications were extracted and eligible for this meta‐analysis. The predominant donor site was the lower extremity (56%), with the anterolateral thigh flap as the major workhorse (30%). The summarized rate of total and partial flap loss was 6.0% (95% CI = 3.0–9.0%, PQ = 1.0000) and 8.0% (95% CI = 6.0–13.0%, PQ = 0.9973), respectively. The rate of arterial and venous thrombosis was 5.0% (95% CI = 3.0–9.0%, PQ = 0.9999) and 8.0% (95% CI = 9.0–37.0%, PQ = 0.9804), respectively. Complication rates of recipient site were: early infection with 7.0% (95% CI = 4.0–11.0%, PQ = 0.9788), hematoma with 6.0% (95% CI = 3.0–8.0%, PQ = 1.0000), seroma with 6.0% (95% CI = 4.0–10.0%, PQ = 0.9948), dehiscence with 7.0% (95% CI = 4.0–11.0%, PQ = 0.9988), and dysesthesia with 6% (95% CI = 4.0–10.0%, PQ = 0.9988). The rate of donor‐site morbidity was 14% (95% CI = 8.0–21.0%, PQ = 0.0895).
Conclusions
Soft tissue free flaps to the upper extremity have comparable success rates, but higher perioperative complication rates (e.g., partial flap loss, infection, and wound dehiscence) compared to the pooled data of lower extremity reconstruction from existing meta‐analysis.
We present single-molecule, real-time sequencing data obtained from a DNA polymerase performing uninterrupted template-directed synthesis using four distinguishable fluorescently labeled ...deoxyribonucleoside triphosphates (dNTPs). We detected the temporal order of their enzymatic incorporation into a growing DNA strand with zero-mode waveguide nanostructure arrays, which provide optical observation volume confinement and enable parallel, simultaneous detection of thousands of single-molecule sequencing reactions. Conjugation of fluorophores to the terminal phosphate moiety of the dNTPs allows continuous observation of DNA synthesis over thousands of bases without steric hindrance. The data report directly on polymerase dynamics, revealing distinct polymerization states and pause sites corresponding to DNA secondary structure. Sequence data were aligned with the known reference sequence to assay biophysical parameters of polymerization for each template position. Consensus sequences were generated from the single-molecule reads at 15-fold coverage, showing a median accuracy of 99.3%, with no systematic error beyond fluorophore-dependent error rates.
Abstract
Summary
Bulk RNA sequencing studies have demonstrated that human leukocyte antigen (HLA) genes may be expressed in a cell type-specific and allele-specific fashion. Single-cell gene ...expression assays have the potential to further resolve these expression patterns, but currently available methods do not perform allele-specific quantification at the molecule level. Here, we present scHLAcount, a post-processing workflow for single-cell RNA-seq data that computes allele-specific molecule counts of the HLA genes based on a personalized reference constructed from the sample’s HLA genotypes.
Availability and implementation
scHLAcount is available under the MIT license at https://github.com/10XGenomics/scHLAcount.
Supplementary information
Supplementary data are available at Bioinformatics online.
•3014 patients with inhalation injury in the National Burn Repository were analyzed.•Patients with undergoing bronchoscopy at admission show a higher risk for pneumonia.•Increased mortality is seen ...when bronchoscopy is performed.
Inhalation injury is a common complication of thermal trauma. Fiberoptic bronchoscopy (FOB) is regarded as current standard practice in diagnosing and grading inhalation injury. Nonetheless, its predictive value in terms of therapeutic decision-making and clinical outcome is controversial.
Adult burn patients with inhalation injury (InI) were selected from the National Burn Repository of the American Burn Association. Subjects were propensity score pair-matched based on injury severity and grouped based on whether or not FOB had been performed (FOB, CTR, respectively). Mortality, incidence of pneumonia, length of hospitalization, length of ICU stay and dependency on mechanical ventilation were compared between the two groups.
3014 patients were matched in two groups with a mean TBSA of 22.4%. There was no significant difference in carboxyhemoglobin fraction at admission. Patients, who underwent FOB on admission had a significantly increased incidence of pneumonia (p < 0.001), mortality (p < 0.05), length of hospitalization (p = 0.002), ICU stay (p < 0.001) and duration of mechanical ventilation (p = 0.006). In a subgroup analysis of patients with TBSA of at least 20%, incidence of pneumonia was significantly higher in the FOB group (p < 0.001) and longer mechanical ventilation was required (p = 0.036).
Diagnosis and grading of InI through FOB is the current standard, although its predictive value regarding key outcome parameters and therapeutic decision-making, remains unclear. The potential procedural risk of FOB itself should be considered. This study demonstrates correlations of FOB with major clinical outcomes in both a general collective of burned adults as well as severely burned adults. Although these findings must be interpreted with caution, they may induce further research into potential harm of FOB and critical review of routine diagnostic FOB in suspected inhalation injury in thermally injured patients.
Optical nanostructures have enabled the creation of subdiffraction detection volumes for single-molecule fluorescence microscopy. Their applicability is extended by the ability to place molecules in ...the confined observation volume without interfering with their biological function. Here, we demonstrate that processive DNA synthesis thousands of bases in length was carried out by individual DNA polymerase molecules immobilized in the observation volumes of zero-mode waveguides (ZMWs) in high-density arrays. Selective immobilization of polymerase to the fused silica floor of the ZMW was achieved by passivation of the metal cladding surface using polyphosphonate chemistry, producing enzyme density contrasts of glass over aluminum in excess of 400:1. Yields of single-molecule occupancies of ≈30% were obtained for a range of ZMW diameters (70-100 nm). Results presented here support the application of immobilized single DNA polymerases in ZMW arrays for long-read-length DNA sequencing.
HLA Typing for the Next Generation Mayor, Neema P; Robinson, James; McWhinnie, Alasdair J M ...
PloS one,
05/2015, Letnik:
10, Številka:
5
Journal Article
Recenzirano
Odprti dostop
Allele-level resolution data at primary HLA typing is the ideal for most histocompatibility testing laboratories. Many high-throughput molecular HLA typing approaches are unable to determine the ...phase of observed DNA sequence polymorphisms, leading to ambiguous results. The use of higher resolution methods is often restricted due to cost and time limitations. Here we report on the feasibility of using Pacific Biosciences' Single Molecule Real-Time (SMRT) DNA sequencing technology for high-resolution and high-throughput HLA typing. Seven DNA samples were typed for HLA-A, -B and -C. The results showed that SMRT DNA sequencing technology was able to generate sequences that spanned entire HLA Class I genes that allowed for accurate allele calling. Eight novel genomic HLA class I sequences were identified, four were novel alleles, three were confirmed as genomic sequence extensions and one corrected an existing genomic reference sequence. This method has the potential to revolutionize the field of HLA typing. The clinical impact of achieving this level of resolution HLA typing data is likely to considerable, particularly in applications such as organ and blood stem cell transplantation where matching donors and recipients for their HLA is of utmost importance.
The mechanical properties of DNA over segments comparable to the size of a protein-binding site (3-10 nm) are examined using an electric-field-induced translocation of single molecules through a ...nanometer diameter pore. DNA, immersed in an electrolyte, is forced through synthetic pores ranging from 0.5 to 1.5 nm in radius in a 10 nm thick Si(3)N(4) membrane using an electric field. To account for the stretching and bending, we use molecular dynamics to simulate the translocation. We have found a threshold for translocation that depends on both the dimensions of the pore and the applied transmembrane bias. The voltage threshold coincides with the stretching transition that occurs in double-stranded DNA near 60 pN.
Background
Compound flaps offer the advantage of one stage defect reconstruction respecting all relevant tissues and early functional recovery by optimal vascularity of all components. Due to its ...specific vascular anatomy and the three‐dimensional donor site, compound flaps with bone components may result in higher complication rates compared to soft tissue compound flaps. The meta‐analysis summarizes the available evidence and evaluates whether bone components are a risk factor for periprocedural complications in upper extremity multidimensional defect reconstruction.
Method
PubMed and Embase were searched for all publications addressing compound free flaps for upper extremity defect reconstruction with bone or soft tissue components published between January 1988 and May 2018. The methodological quality was assessed with the American Society of Plastic Surgeons Evidence Rating Scale for Therapeutic Studies. Flap loss, thrombosis rate, early infection, hematoma, seroma, as well as donor site complications were extracted and analyzed.
Results
Twelve out of 1157 potentially eligible studies (evidence‐III) comprising 159 patients were finally included with publication bias for all summarized complication rates. Complication rates for flaps with/ without bone components were: total flap loss 5%, 95% CI = 3%–10% (6%/5%); partial flap loss 8%, 95% CI = 5%–15%, (9%/8%); arterial/venous thrombosis 7%, 95% CI = 4%–12%, (8%/5%)/14%, 95% CI = 9%–21% (16%/6%, P < .05) with higher risk for flaps with bone components; infection 6%, 95% CI = 3%–12% (6%/6%); hematoma 6%, 95% CI = 3%–11% (6%/5%); seroma 5%, 95% CI = 3%–10% (5%/5%); dehiscence 10%, 95% CI = 6%–17% (11%/9%).
Conclusion
Compound flaps for upper extremity defect reconstruction including bone components have a higher venous thrombosis rate compared to compound soft‐tissue flaps.
Recent pan-genome studies have revealed an abundance of DNA sequences in human genomes that are not present in the reference genome. A lion's share of these non-reference sequences (NRSs) cannot be ...reliably assembled or placed on the reference genome. Improvements in long-read and synthetic long-read (aka linked-read) technologies have great potential for the characterization of NRSs. While synthetic long reads require less input DNA than long-read datasets, they are algorithmically more challenging to use. Except for computationally expensive whole-genome assembly methods, there is no synthetic long-read method for NRS detection. We propose a novel integrated alignment-based and local assembly-based algorithm, Novel-X, that uses the barcode information encoded in synthetic long reads to improve the detection of such events without a whole-genome de novo assembly. Our evaluations demonstrate that Novel-X finds many non-reference sequences that cannot be found by state-of-the-art short-read methods. We applied Novel-X to a diverse set of 68 samples from the Polaris HiSeq 4000 PGx cohort. Novel-X discovered 16 691 NRS insertions of size > 300 bp (total length 18.2 Mb). Many of them are population specific or may have a functional impact.
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