Next-generation sequencing platforms are powerful technologies, providing gigabases of genetic information in a single run. An important prerequisite for high-throughput DNA sequencing is the development of robust and cost-effective preprocessing protocols for DNA sample library construction. Here we report the development of a semi-automated sample preparation protocol to produce adaptor-ligated fragment libraries. Using a liquid-handling robot in conjunction with Carboxy Terminated Magnetic Beads, we labeled each library sample using a unique 6 bp DNA barcode, which allowed multiplex sample processing and sequencing of 32 libraries in a single run using Applied Biosystems' SOLiD sequencer. We applied our semi-automated pipeline to targeted medical resequencing of nuclear candidate genes in individuals affected by mitochondrial disorders. This novel method is capable of preparing as much as 32 DNA libraries in 2.01 days (8-hour workday) for emulsion PCR/high throughput DNA sequencing, increasing sample preparation production by 8-fold.
Sample preparation for Roche/454, ABI/SOLiD and Life Technologies/Ion Torrent sequencing are based on amplification of library fragments on the surface of beads prior to sequencing. Commonly, libraries are barcoded and pooled, to maximise the sequence output of each sequence run. Here, we describe a novel approach for normalization of multiplex next generation sequencing libraries after emulsion PCR. Briefly, amplified libraries carrying unique barcodes are prepared by fluorescent tagging of complementary sequences and then resolved by high-speed flow cytometric sorting of labeled emulsion PCR beads. The protocol is simple and provides an even sequence distribution of multiplex libraries when sequencing the flow-sorted beads. Moreover, since many empty and mixed emulsion PCR beads are removed, the approach gives rise to a substantial increase in sequence quality and mean read length, as compared to that obtained by standard enrichment protocols.
A set of 96 molecular barcode adaptors specifically designed for the SOLiD™ platform have been validated for use with DNA fragment and paired end libraries. Moreover, the barcode system is adapted for multiplexed Serial Analysis of Gene Expression (SAGE). DNA libraries are constructed with a multiplex adaptor which consists of three segments: (1) an internal sequencing primer binding site, (2) a barcode decamer sequence and (3) a P2 PCR priming site. The barcode and target DNA are then sequenced as two separate reads from the same strand allowing for the libraries to be pooled in a multiplexed emulsion PCR and deposited into a single spot on a SOLiD™ slide. Similarly, SAGE libraries are constructed with a modified adaptor allowing for the addition of unique barcode primers with a short cycle amplification consistent with the SOLiD™ barcoding system. The modular barcoding design requires only 5bp of sequencing to distinguish 16-plex samples and 10bp of sequencing to distinguish 96-plex samples. The barcodes are optimized in sets of four wherein each set is color balanced at every position. Importantly, clear discrimination between barcode samples is achieved by maintaining a minimum Hamming distance of 3 colorspace calls for optimal data integrity. The DNA barcode system was validated by sequencing of E. coli fragment libraries. Error rates and quality value (QV) scores for the barcode reads were found to be consistent across the final set. Importantly, QV scores were also consistent for the reads, indicating minimal effects of the barcode decamers on bead templating and ligation sequencing efficiency. Furthermore, the set of 16 SAGE barcoded samples yielded Pearson correlations above 0.98. Ongoing development studies include integration with methods of target enrichment that will further enable high levels of DNA and RNA expression library multiplexing afforded by the increasing throughput of the SOLiD™ system.
Patients with a personal or familial history of thromboembolism are considered at higher risk for thromboembolic disease after knee arthroplasty. While it remains unclear why some patients develop deep vein thrombosis (DVT) or pulmonary embolism (PE) despite similar operative procedures and the same prophylactic regimen, we presume one explanation would be genetic predisposition.
We determined the frequency of 12 factors including antithrombin III activity, prothrombin gene mutations, and the presence of phospholipid antibodies in a high-risk patient cohort and compared those findings with the known prevalence in the population at large.
Patients and Methods
Patients identified preoperatively as having a personal or familial history of DVT and/or PE were referred for hemostatic serum and genetic tests, including % antithrombin III activity (ATIII), protein C and protein S activities, APC resistance, Factor V gene (Leiden) mutations, prothrombin gene mutations, lupus anticoagulant antibody presence, cardiolipin antibody presence, phosphatidyl antibody presence, β2-glycoprotein antibody presence, and serum homocysteine and lipoprotein(a) levels The frequencies of varying abnormalities were identified and compared to the prevalence reported in the literature.
Forty-three of 1944 patients undergoing knee arthroplasty had a history of DVT or PE. Sixteen of 43 (37%) patients had an abnormality and eight of these (19%) had two or more abnormalities. The frequency of nine of the 12 tests appeared to be greater in this cohort than in the population at large.
Patients with a personal or familial history of DVT or PE appear to have a high frequency of hereditary prothrombotic abnormalities. Preoperative evaluation by a hematologist may be warranted in patients with a personal or familial history of DVT or PE as the postoperative anticoagulation protocols may be altered and identification of these abnormalities may affect a patient’s risk for other disease states.
Level of Evidence
Level IV, diagnostic study. See Guidelines for Authors for a complete description of levels of evidence.
Clinically silent deep vein thrombosis (DVT) is common and may cause chronic venous disease that resembles post-thrombotic syndrome.
We evaluated whether peripheral venous disease in a general population shares risk factors with DVT.
In an established cohort of 2,404 men and women, the San Diego Population Study, peripheral venous disease was evaluated using physical exam, symptom assessment, and venous ultrasound. We performed a case control study including 308 cases in 4 hierarchical groups by severity, and 346 controls without venous abnormalities, frequency matched to cases by 10-year age group, race and sex. Cases and controls had no prior history of venous thrombosis. Hemostatic risk factors were measured in cases and controls.
Accounting for age, obesity and family history of leg ulcer, ORs for elevated factor VIII, von Willebrand factor, D-dimer, and for factor V Leiden were 1.4 (95% CI 0.9–2.1), 1.5 (CI 1.0–2.3), 1.7 (CI 1.1–2.8), and 1.1 (CI 0.5–2.4), respectively. These associations were larger in the two most severe case groups; ORs 2.0 (CI 1.0–3.8), 1.7 (CI 0.9–3.3), 2.7 (CI 1.2–6.1) and 2.3 (CI 0.8–7.1). Each hemostatic factor was also associated with severity of venous disease, for example elevated D-dimer was associated with a 2.2-fold increased odds of being in one higher severity group. Prothrombin 20210A was not associated with venous disease.
DVT risk factors are associated with presence and severity of peripheral venous disease. Results support a hypothesis that peripheral venous disease may sometimes be post-thrombotic syndrome due to previous unrecognized DVT.
deep vein thrombosis; venous insufficiency; risk factors; epidemiology; blood coagulation
Massively parallel DNA sequencing, when combined with sequence capture methodologies, reduces the time, cost, and effort required to interrogate multiple genomic loci. We have demonstrated that high levels of multiplexing are achievable by combining HybSelect parallel sample processing capabilities with bar-coding reagents. After automated sequence capture with ™, targeted next-generation sequencing (tNGS) was carried out on the ™ Sequencing System. The use of barcodes on the sequencing adaptors allows the confident identification and tracking of samples, and greatly strengthens traditional LIMS (Laboratory Information Management Systems) in regulated production sequencing environments. Bar-coded targets from hundreds of individuals can be sequenced per week by integrating the workflows of the SOLiD System with a febit HybSelect instrument. We have applied this process to several human disease related targets and will present results from first studies.
The multiplexing becomes the major limitation of the next-generation sequencing (NGS) in application to low complexity samples. Physical space segregation allows limited multiplexing, while the existing barcode approach only permits simultaneously analysis of up to several dozen samples.
Here we introduce pair-barcode sequencing (PBS), an economic and flexible barcoding technique that permits parallel analysis of large-scale multiplexed samples. In two pilot runs using SOLiD sequencer (Applied Biosystems Inc.), 32 independent pair-barcoded miRNA libraries were simultaneously discovered by the combination of 4 unique forward barcodes and 8 unique reverse barcodes. Over 174,000,000 reads were generated and about 64% of them are assigned to both of the barcodes. After mapping all reads to pre-miRNAs in miRBase, different miRNA expression patterns are captured from the two clinical groups. The strong correlation using different barcode pairs and the high consistency of miRNA expression in two independent runs demonstrates that PBS approach is valid.
By employing PBS approach in NGS, large-scale multiplexed pooled samples could be practically analyzed in parallel so that high-throughput sequencing economically meets the requirements of samples which are low sequencing throughput demand.
barcode; next-generation sequencing; miRNA; breast cancer
Compared to classical genotyping, targeted next-generation sequencing (tNGS) can be custom-designed to interrogate entire genomic regions of interest, in order to detect novel as well as known variants. To bring down the per-sample cost, one approach is to pool barcoded NGS libraries before sample enrichment. Still, we lack a complete understanding of how this multiplexed tNGS approach and the varying performance of the ever-evolving analytical tools can affect the quality of variant discovery. Therefore, we evaluated the impact of different software tools and analytical approaches on the discovery of single nucleotide polymorphisms (SNPs) in multiplexed tNGS data. To generate our own test model, we combined a sequence capture method with NGS in three experimental stages of increasing complexity (E. coli genes, multiplexed E. coli, and multiplexed HapMap BRCA1/2 regions).
We successfully enriched barcoded NGS libraries instead of genomic DNA, achieving reproducible coverage profiles (Pearson correlation coefficients of up to 0.99) across multiplexed samples, with <10% strand bias. However, the SNP calling quality was substantially affected by the choice of tools and mapping strategy. With the aim of reducing computational requirements, we compared conventional whole-genome mapping and SNP-calling with a new faster approach: target-region mapping with subsequent ‘read-backmapping’ to the whole genome to reduce the false detection rate. Consequently, we developed a combined mapping pipeline, which includes standard tools (BWA, SAMtools, etc.), and tested it on public HiSeq2000 exome data from the 1000 Genomes Project. Our pipeline saved 12 hours of run time per Hiseq2000 exome sample and detected ~5% more SNPs than the conventional whole genome approach. This suggests that more potential novel SNPs may be discovered using both approaches than with just the conventional approach.
We recommend applying our general ‘two-step’ mapping approach for more efficient SNP discovery in tNGS. Our study has also shown the benefit of computing inter-sample SNP-concordances and inspecting read alignments in order to attain more confident results.
Two-stage mapping; Read-backmapping; Software performance; SNP discovery; Multiplexed targeted next-generation sequencing
Improved medical treatment options have advanced pediatric care but often necessitate both invasive vascular procedures and venous access predisposing these patients to venous thrombotic events. Although pediatric deep vein thrombosis (DVT) is an increasingly recognized phenomenon, high-quality evidence for its antithrombotic treatment in general remains limited, and even more so with respect to thrombolytic therapy. Correspondingly, current American College of Chest Physicians guidelines discourage the routine use of thrombolytic therapy for pediatric DVT; by contrast, American Heart Association guidelines suggest consideration for such therapy in young patients in whom the balance of benefit to risk may be most favorable. The developing hemostatic system and relative rarity of thrombotic events have historically posed impediments to the design and conduction of prospective clinical trials of thrombolysis in children. This narrative review summarizes available information regarding thrombolytic therapy for pediatric DVT.
thrombolysis; deep vein thrombosis; pediatric
Background: Deep vein thrombosis (DVT) is one of the most common complications of total hip (THA) and total knee arthroplasty (TKA). Though the reported incidence of DVT is very high, that of proximal DVT is low and that of fatal thromboembolism is very low. Hence the issue of prophylaxis for DVT remains controversial.
The incidence of DVT is based on various studies in European and American populations. The Asian population is genetically and socially quite different from American and European populations, and the incidence of DVT can be quite different. Therefore a prospective study was initiated at our centre to determine incidence of DVT after THA and TKA in Indian patients.
Methods: A prospective study was conducted on 60 hips in 45 patients and 46 knees in 26 patients who underwent THA and TKA respectively, without any known risk factors for thromboembolic disease. DVT was studied by preoperative and postoperative serial colour Doppler ultrasonography. No prophylaxis was given to any of the patients.
Results: DVT was found in two patients who had undergone THA. No case of DVT was detected in any patient who had undergone TKA.
Conclusion: These results suggest that the incidence of DVT in Indian patients is very low and is not comparable with American and European populations. It is therefore not cost effective to advise prophylaxis in Indian patients undergoing THA/TKA who have no known risk factors for DVT.
Multilocus sequence typing (MLST) is a widely used system for typing microorganisms by sequence analysis of housekeeping genes. The main advantage of MLST in comparison to other typing techniques is the unambiguity and transferability of sequence data. However, a main disadvantage is the high cost of DNA sequencing. Here we introduce a high-throughput MLST (HiMLST) method that employs next-generation sequencing (NGS) technology (Roche 454), to generate large quantities of high-quality MLST data at low costs. The HiMLST protocol consists of two steps. In the first step MLST target genes are amplified by PCR in multi-well plates. During this PCR the amplicons of each bacterial isolate are provided with a unique DNA barcode, the multiplex identifier (MID). In the second step all amplicons are pooled and sequenced in a single NGS-run. The MLST profile of each individual isolate can be retrieved easily using its unique MID. With HiMLST we have profiled 575 isolates of Legionella pneumophila, Staphylococcus aureus, Pseudomonas aeruginosa and Streptococcus pneumoniae in mixed species HiMLST experiments. In conclusion, the introduction of HiMLST paves the way for a broad employment of the MLST as a high-quality and cost-effective method for typing microbial species.
Deep vein thrombosis (DVT) is caused by obstruction of blood flow of deep veins in upper and lower limb. One of the precipitating factors for DVT is surgery under general anesthesia exceeding 30 min. However, there are very few reports of DVT associated with surgery of oral and maxillofacial region. In this paper we report two cases of DVT involving left ilio-femoropopliteal deep vein in one patient treated for fractured left angle of mandible and left peroneal vein in the other patient treated for oral sub mucous fibrosis. Clinical and color Doppler examination were performed to diagnose the condition and were referred to vascular surgical unit of higher institute for further management. These cases illustrates any surgery of maxillofacial region is not free from risk of DVT, which can cause fatal pulmonary thromboembolism.
Deep vein thrombosis; fracture mandible; oral and maxillofacial surgery; oral submucous fibrosis; pulmonary embolism; venous thromboembolism
The costs and efforts for sample preparation of hundreds of individuals, their genomic enrichment for regions of interest, and sufficient deep sequencing bring a significant burden to next-generation sequencing-based experiments. We investigated whether pooling of samples at the level of genomic DNA would be a more versatile strategy for lowering the costs and efforts for common disease-associated rare variant detection in candidate genes or associated loci in a substantial patient cohort. We performed a pilot experiment using five pools of 20 abdominal aortic aneurysm (AAA) patients that were enriched on separate microarrays for the reported 9p21.3 associated locus and 42 additional AAA candidate genes, and sequenced on the SOLiD platform. Here, we discuss challenges and limitations connected to this approach and show that the high number of novel variants detected per pool and allele frequency deviations to the usually highly false positive cut-off region for variant detection in non-pooled samples can be limiting factors for successful variant prioritization and confirmation. We conclude that barcode indexing of individual samples before pooling followed by a multiplexed enrichment strategy should be preferred for detection of rare genetic variants in larger sample sets rather than a genomic DNA pooling strategy.
Abdominal aortic aneurysm; Common disease; Rare variants; Targeted genomic enrichment; Genomic DNA pooling; SOLiD next-generation sequencing
Aortopathies are a group of disorders characterized by aneurysms, dilation, and tortuosity of the aorta. Because of the phenotypic overlap and genetic heterogeneity of diseases featuring aortopathy, molecular testing is often required for timely and correct diagnosis of affected individuals. In this setting next generation sequencing (NGS) offers several advantages over traditional molecular techniques.
The purpose of our study was to compare NGS enrichment methods for a clinical assay targeting the nine genes known to be associated with aortopathy. RainDance emulsion PCR and SureSelect RNA-bait hybridization capture enrichment methods were directly compared by enriching DNA from eight samples. Enriched samples were barcoded, pooled, and sequenced on the Illumina HiSeq2000 platform. Depth of coverage, consistency of coverage across samples, and the overlap of variants identified were assessed. This data was also compared to whole-exome sequencing data from ten individuals.
Read depth was greater and less variable among samples that had been enriched using the RNA-bait hybridization capture enrichment method. In addition, samples enriched by hybridization capture had fewer exons with mean coverage less than 10, reducing the need for followup Sanger sequencing. Variants sets produced were 77% concordant, with both techniques yielding similar numbers of discordant variants.
When comparing the design flexibility, performance, and cost of the targeted enrichment methods to whole-exome sequencing, the RNA-bait hybridization capture enrichment gene panel offers the better solution for interrogating the aortopathy genes in a clinical laboratory setting.
Aortopathy; Hybridization capture; Marfan syndrome; Next generation sequencing (NGS); Target enrichment; Emulsion PCR
Motivation: Next-generation sequencing technologies have enabled the sequencing of several human genomes in their entirety. However, the routine resequencing of complete genomes remains infeasible. The massive capacity of next-generation sequencers can be harnessed for sequencing specific genomic regions in hundreds to thousands of individuals. Sequencing-based association studies are currently limited by the low level of multiplexing offered by sequencing platforms. Pooled sequencing represents a cost-effective approach for studying rare variants in large populations. To utilize the power of DNA pooling, it is important to accurately identify sequence variants from pooled sequencing data. Detection of rare variants from pooled sequencing represents a different challenge than detection of variants from individual sequencing.
Results: We describe a novel statistical approach, CRISP [Comprehensive Read analysis for Identification of Single Nucleotide Polymorphisms (SNPs) from Pooled sequencing] that is able to identify both rare and common variants by using two approaches: (i) comparing the distribution of allele counts across multiple pools using contingency tables and (ii) evaluating the probability of observing multiple non-reference base calls due to sequencing errors alone. Information about the distribution of reads between the forward and reverse strands and the size of the pools is also incorporated within this framework to filter out false variants. Validation of CRISP on two separate pooled sequencing datasets generated using the Illumina Genome Analyzer demonstrates that it can detect 80–85% of SNPs identified using individual sequencing while achieving a low false discovery rate (3–5%). Comparison with previous methods for pooled SNP detection demonstrates the significantly lower false positive and false negative rates for CRISP.
Availability: Implementation of this method is available at http://polymorphism.scripps.edu/∼vbansal/software/CRISP/
Lower limb cellulitis and deep vein thrombosis share clinical features and investigation of patients with cellulitis for concurrent DVT is common. The prevalence of DVT in this group is uncertain. This study aimed to determine the prevalence of deep vein thrombosis (DVT) in patients with lower limb cellulitis and to investigate the utility of applying the Wells algorithm to this patient group.
Patients admitted with lower limb cellulitis prospectively underwent a likelihood assessment for DVT using the Wells criteria followed by investigation with D-dimer and ultrasonography of ipsilateral femoral veins as appropriate. Diagnoses of contralateral DVT or pulmonary embolism during admission were recorded.
200 patients assessed for DVT. 20% of subjects were high risk by Wells criteria. D-dimer was elevated in 74% and 79% underwent insonation of the affected leg. Ipsilateral DVT was found in 1 patient (0.5%) and non-ipsilateral VTE in a further 2 (1%).
Deep vein thrombosis rarely occurs concurrently with lower limb cellulitis. The Wells score substantially overestimates the likelihood of DVT due to an overlap of clinical signs. Investigation for DVT in patients with cellulitis is likely to yield few diagnoses and is not warranted in the absence of a hypercoaguable state.
ACTRN: 12610000792022 (https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=320662)
Cellulitis; Erysipelas; Venous thromboembolism; Venous thrombosis
Deep vein thrombosis (DVT) is a major health problem that requires improved prophylaxis and treatment. Inflammatory conditions such as infection, cancer and autoimmune diseases are risk factors for DVT. We and others have recently shown that extracellular DNA fibers produced in inflammation and known as neutrophil extracellular traps (NETs) contribute to experimental DVT. NETs stimulate thrombus formation and coagulation and are abundant in thrombi in animal models of DVT. It appears that, in addition to fibrin and VWF, NETs represent a third thrombus scaffold. Here we review how NETs stimulate thrombosis and discuss known and potential interactions of NETs with endothelium, platelets, red blood cells, coagulation factors and how NETs could influence thrombolysis. We propose that drugs which inhibit NET formation or facilitate NET degradation may prevent or treat DVT.
deep vein thrombosis; inflammation; mouse model; NETs; DNase
Deep vein thrombosis initiation is mediated by cross talk between monocytes, neutrophils, and platelets.
Deep vein thrombosis (DVT) is a major cause of cardiovascular death. The sequence of events that promote DVT remains obscure, largely as a result of the lack of an appropriate rodent model. We describe a novel mouse model of DVT which reproduces a frequent trigger and resembles the time course, histological features, and clinical presentation of DVT in humans. We demonstrate by intravital two-photon and epifluorescence microscopy that blood monocytes and neutrophils crawling along and adhering to the venous endothelium provide the initiating stimulus for DVT development. Using conditional mutants and bone marrow chimeras, we show that intravascular activation of the extrinsic pathway of coagulation via tissue factor (TF) derived from myeloid leukocytes causes the extensive intraluminal fibrin formation characteristic of DVT. We demonstrate that thrombus-resident neutrophils are indispensable for subsequent DVT propagation by binding factor XII (FXII) and by supporting its activation through the release of neutrophil extracellular traps (NETs). Correspondingly, neutropenia, genetic ablation of FXII, or disintegration of NETs each confers protection against DVT amplification. Platelets associate with innate immune cells via glycoprotein Ibα and contribute to DVT progression by promoting leukocyte recruitment and stimulating neutrophil-dependent coagulation. Hence, we identified a cross talk between monocytes, neutrophils, and platelets responsible for the initiation and amplification of DVT and for inducing its unique clinical features.
Background and Objectives:
Deep vein thrombosis (DVT) occurs at a lower rate in Asia than in the rest of the world. We wanted to study the significance and efficacy of low molecular weight heparin (LMWH) in prophylaxis of DVT in major general surgical patients in the Kashmir Valley (India, Asia) so as to make it a routine in our patients.
Patients and Methods:
This was a prospective study in which the effect of LMWH was compared with no prophylaxis.
LMWHs are more effective than no prophylaxis in the prevention of DVT and pulmonary thromboembolism in highest-risk general surgical patients (odds ratio = 16.64; 95% confidence interval = 3.63–1130.03; P-value = 0.014).
LMWHs have a significant prophylactic effect on DVT in general surgical patients, with a higher benefit to risk ratio, and, in spite of the low incidence of DVT in Asia, its prophylaxis should routinely be considered in this part of the world as well, preferably in the form of LMWHs.
Heparin; prophylaxis; thrombosis
Hyperhomocysteinemia is a known risk factor for the development of deep vein thrombosis (DVT). Various studies have been conducted in the western countries to know the prevalence of hyperhomocysteinemia in patients with DVT and in general population. There is no documented literature of the prevalence of hyperhomocysteinemia in Indian population. Thus the aim of this study was to determine the prevalence of hyperhomocysteinemia in cases of DVT in our population. To evaluate the prevalence of hyperhomocysteinemia, a prospective cross sectional study done on a total of 70 patients admitted in KLES Dr Prabhakar Kore hospital, Belgaum, India. DVT was confirmed by Doppler examination. Serum homocysteine was measured and the data analysed. Statistical significance was calculated using chi square test. A total of 70 patients were studied of which 53 were males and 17 were females. The prevalence of hyperhomocysteinemia among the cases of DVT was 31.428%.The prevalence among males was 35.85% and among females was 17.64%.There was statistically significant association between hyperhomocysteinemia and presence of ischaemic heart disease with a p value of 0.005 on chi square analysis. The prevalence of hyperhomocysteinemia in cases of deep vein thrombosis in our population was 31.428%. There was a statistically significant association between hyperhomocysteinemia and ischaemic heart disease.
Hyperhomocyteinemia; Deep vein thrombosis
Objective. To evaluate the incidence of deep vein thrombosis in hospitalized Chinese medical patients and the impact of DVT prophylaxis. Methods. All cases of confirmed proximal DVT from 1 January 2005 to 31 December 2008 were reviewed retrospectively to determine the presence of risk factors and whether DVT developed: during hospitalization in medical wards or in case of readmission with a diagnosis of DVT within 14 days of discharge from a recent admission to medical wards. The impact of prophylaxis will be estimated by comparing the annual incidence of proximal DVT among medical patients hospitalized from 2005 to 2007 with that of 2008 (DVT prophylaxis commonly used). Results. From 1 January 2005 to 31 December 2008, 3938 Doppler ultrasound studies were performed for suspected DVT. Proximal DVT was diagnosed in 687 patients. The calculated incidence of proximal DVT among medical patients hospitalized for at least two days was 1.8%, 2%, and 1.7% for the year 2005, 2006, and 2007, respectively. The incidence was 1.1% for 2008 (P < .001). Conclusion. Proximal DVT was substantial in Chinese medical patients, and DVT prophylaxis might reduce such risk.
Air travel has been linked with the development of deep vein thrombosis (DVT) since the 1950s with a number of plausible explanations put forward for causation. No systematic review of the literature exploring this association has previously been published.
A comprehensive search was undertaken (Data bases searched were: MEDLINE, EMBASE, Cochrane Library) for studies that estimated both the incidence and the risk of DVT in air travellers relative to non-air travellers.
In total 254 studies were identified but only six incidence studies and four risk studies met inclusion criteria justifying their use in a systematic review. Incidence of symptomatic DVT ranged from (0%) in one study to (0.28%) which was reported in pilots over ten years. The incidence of asymptomatic DVT ranged from (0%) to (10.34%). Pooled odds ratios for the two case control studies examining the risk of DVT following air travel were 1.11 (95% CI: 0.64–1.94). Pooled odds ratios for all models of travel including two studies of prolonged air travel (more than three hours) were 1.70 (95% CI: 0.89–3.22).
We found no definitive evidence that prolonged (more than 3-hours) travel including air travel, increases the risk of DVT. There is evidence to suggest that flights of eight hours or more increase the risk of DVT if additional risk factors exist.
May-Thurner syndrome is a rarely diagnosed condition in which patients develop iliofemoral deep venous thrombosis (DVT) due to an anatomical variant in which the right common iliac artery overlies and compresses the left common iliac vein against the lumbar spine. This variant has been shown to be present in over 20% of the population; however, it is rarely considered in the differential diagnosis of DVT, particularly in patients with other risk factors. Systemic anticoagulation alone is insufficient treatment, and a more aggressive approach is necessary to prevent recurrent DVT. Herein, we present a patient with multiple risk factors for DVT. With a comprehensive diagnostic approach, she was found to have May-Thurner syndrome. Local infusion of thrombolytics as well as mechanical thrombectomy failed to resolve the thrombus. Subsequently the patient underwent successful stent placement in the area that was compressed followed by 6 months of chronic anticoagulation with warfarin. There has been no recurrence of DVT in the ensuing 18 months.
Primer design for highly variable DNA sequences is difficult, and experimental success requires attention to many interacting constraints. The advent of next-generation sequencing methods allows the investigation of rare variants otherwise hidden deep in large populations, but requires attention to population diversity and primer localization in relatively conserved regions, in addition to recognized constraints typically considered in primer design.
Design constraints include degenerate sites to maximize population coverage, matching of melting temperatures, optimizing de novo sequence length, finding optimal bio-barcodes to allow efficient downstream analyses, and minimizing risk of dimerization. To facilitate primer design addressing these and other constraints, we created a novel computer program (PrimerDesign) that automates this complex procedure. We show its powers and limitations and give examples of successful designs for the analysis of HIV-1 populations.
PrimerDesign is useful for researchers who want to design DNA primers and probes for analyzing highly variable DNA populations. It can be used to design primers for PCR, RT-PCR, Sanger sequencing, next-generation sequencing, and other experimental protocols targeting highly variable DNA samples.
Primer design; DNA sequencing; Amplicon sequencing; Next-generation sequencing; PCR; Primer dimer; Bio-barcodes; Multiplex
The characterization of bacterial communities using DNA sequencing has revolutionized our ability to study microbes in nature and discover the ways in which microbial communities affect ecosystem functioning and human health. Here we describe Serial Illumina Sequencing (SI-Seq): a method for deep sequencing of the bacterial 16S rRNA gene using next-generation sequencing technology. SI-Seq serially sequences portions of the V5, V6 and V7 hypervariable regions from barcoded 16S rRNA amplicons using an Illumina short-read genome analyzer. SI-Seq obtains taxonomic resolution similar to 454 pyrosequencing for a fraction of the cost, and can produce hundreds of thousands of reads per sample even with very high multiplexing. We validated SI-Seq using single species and mock community controls, and via a comparison to cystic fibrosis lung microbiota sequenced using 454 FLX Titanium. Our control runs show that SI-Seq has a dynamic range of at least five orders of magnitude, can classify >96% of sequences to the genus level, and performs just as well as 454 and paired-end Illumina methods in estimation of standard microbial ecology diversity measurements. We illustrate the utility of SI-Seq in a pilot sample of central airway secretion samples from cystic fibrosis patients.