An integrated map of structural variation in 2,504 human genomes.

Structural variants are implicated in numerous diseases and make up the majority of varying nucleotides among human genomes. Here we describe an integrated set of eight structural variant classes comprising both balanced and unbalanced variants, which we constructed using short-read DNA sequencing data and statistically phased onto haplotype blocks in 26 human populations. Analysing this set, we identify numerous gene-intersecting structural variants exhibiting population stratification and describe naturally occurring homozygous gene knockouts that suggest the dispensability of a variety of human genes. We demonstrate that structural variants are enriched on haplotypes identified by genome-wide association studies and exhibit enrichment for expression quantitative trait loci. Additionally, we uncover appreciable levels of structural variant complexity at different scales, including genic loci subject to clusters of repeated rearrangement and complex structural variants with multiple breakpoints likely to have formed through individual mutational events. Our catalogue will enhance future studies into structural variant demography, functional impact and disease association.

Tracy: basecalling, alignment, assembly and deconvolution of sanger chromatogram trace files.

BACKGROUND: DNA sequencing is at the core of many molecular biology laboratories. Despite its long history, there is a lack of user-friendly Sanger sequencing data analysis tools that can be run interactively as a web application or at large-scale in batch from the command-line. RESULTS: We present Tracy, an efficient and versatile command-line application that enables basecalling, alignment, assembly and deconvolution of sequencing chromatogram files. Its companion web applications make all functionality of Tracy easily accessible using standard web browser technologies and interactive graphical user interfaces. Tracy can be easily integrated in large-scale pipelines and high-throughput settings, and it uses state-of-the-art file formats such as JSON and BCF for reporting chromatogram sequencing results and variant calls. The software is open-source and freely available at https://github.com/gear-genomics/tracy, the companion web applications are hosted at https://www.gear-genomics.com. CONCLUSIONS: Tracy can be routinely applied in large-scale validation efforts conducted in clinical genomics studies as well as for high-throughput genome editing techniques that require a fast and rapid method to confirm discovered variants or engineered mutations. Molecular biologists benefit from the companion web applications that enable installation-free Sanger chromatogram analyses using intuitive, graphical user interfaces.

Alfred: interactive multi-sample BAM alignment statistics, feature counting and feature annotation for long- and short-read sequencing.

SUMMARY: Harmonizing quality control (QC) of large-scale second and third-generation sequencing datasets is key for enabling downstream computational and biological analyses. We present Alfred, an efficient and versatile command-line application that computes multi-sample QC metrics in a read-group aware manner, across a wide variety of sequencing assays and technologies. In addition to standard QC metrics such as GC bias, base composition, insert size and sequencing coverage distributions it supports haplotype-aware and allele-specific feature counting and feature annotation. The versatility of Alfred allows for easy pipeline integration in high-throughput settings, including DNA sequencing facilities and large-scale research initiatives, enabling continuous monitoring of sequence data quality and characteristics across samples. Alfred supports haplo-tagging of BAM/CRAM files to conduct haplotype-resolved analyses in conjunction with a variety of next-generation sequencing based assays. Alfred's companion web application enables interactive exploration of results and comparison to public datasets. AVAILABILITY AND IMPLEMENTATION: Alfred is open-source and freely available at https://tobiasrausch.com/alfred/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Genetic code expansion for multiprotein complex engineering.

We present a baculovirus-based protein engineering method that enables site-specific introduction of unique functionalities in a eukaryotic protein complex recombinantly produced in insect cells. We demonstrate the versatility of this efficient and robust protein production platform, 'MultiBacTAG', (i) for the fluorescent labeling of target proteins and biologics using click chemistries, (ii) for glycoengineering of antibodies, and (iii) for structure-function studies of novel eukaryotic complexes using single-molecule Förster resonance energy transfer as well as site-specific crosslinking strategies.

Assembly and diploid architecture of an individual human genome via single-molecule technologies.

We present the first comprehensive analysis of a diploid human genome that combines single-molecule sequencing with single-molecule genome maps. Our hybrid assembly markedly improves upon the contiguity observed from traditional shotgun sequencing approaches, with scaffold N50 values approaching 30 Mb, and we identified complex structural variants (SVs) missed by other high-throughput approaches. Furthermore, by combining Illumina short-read data with long reads, we phased both single-nucleotide variants and SVs, generating haplotypes with over 99% consistency with previous trio-based studies. Our work shows that it is now possible to integrate single-molecule and high-throughput sequence data to generate de novo assembled genomes that approach reference quality.

Primate genome architecture influences structural variation mechanisms and functional consequences.

Although nucleotide resolution maps of genomic structural variants (SVs) have provided insights into the origin and impact of phenotypic diversity in humans, comparable maps in nonhuman primates have thus far been lacking. Using massively parallel DNA sequencing, we constructed fine-resolution genomic structural variation maps in five chimpanzees, five orang-utans, and five rhesus macaques. The SV maps, which are comprised of thousands of deletions, duplications, and mobile element insertions, revealed a high activity of retrotransposition in macaques compared with great apes. By comparison, nonallelic homologous recombination is specifically active in the great apes, which is correlated with architectural differences between the genomes of great apes and macaque. Transcriptome analyses across nonhuman primates and humans revealed effects of species-specific whole-gene duplication on gene expression. We identified 13 gene duplications coinciding with the species-specific gain of tissue-specific gene expression in keeping with a role of gene duplication in the promotion of diversification and the acquisition of unique functions. Differences in the present day activity of SV formation mechanisms that our study revealed may contribute to ongoing diversification and adaptation of great ape and Old World monkey lineages.

CGGBP1 mitigates cytosine methylation at repetitive DNA sequences.

BACKGROUND: CGGBP1 is a repetitive DNA-binding transcription regulator with target sites at CpG-rich sequences such as CGG repeats and Alu-SINEs and L1-LINEs. The role of CGGBP1 as a possible mediator of CpG methylation however remains unknown. At CpG-rich sequences cytosine methylation is a major mechanism of transcriptional repression. Concordantly, gene-rich regions typically carry lower levels of CpG methylation than the repetitive elements. It is well known that at interspersed repeats Alu-SINEs and L1-LINEs high levels of CpG methylation constitute a transcriptional silencing and retrotransposon inactivating mechanism. RESULTS: Here, we have studied genome-wide CpG methylation with or without CGGBP1-depletion. By high throughput sequencing of bisulfite-treated genomic DNA we have identified CGGBP1 to be a negative regulator of CpG methylation at repetitive DNA sequences. In addition, we have studied CpG methylation alterations on Alu and L1 retrotransposons in CGGBP1-depleted cells using a novel bisulfite-treatment and high throughput sequencing approach. CONCLUSIONS: The results clearly show that CGGBP1 is a possible bidirectional regulator of CpG methylation at Alus, and acts as a repressor of methylation at L1 retrotransposons.

Efficient storage of high throughput DNA sequencing data using reference-based compression.

Data storage costs have become an appreciable proportion of total cost in the creation and analysis of DNA sequence data. Of particular concern is that the rate of increase in DNA sequencing is significantly outstripping the rate of increase in disk storage capacity. In this paper we present a new reference-based compression method that efficiently compresses DNA sequences for storage. Our approach works for resequencing experiments that target well-studied genomes. We align new sequences to a reference genome and then encode the differences between the new sequence and the reference genome for storage. Our compression method is most efficient when we allow controlled loss of data in the saving of quality information and unaligned sequences. With this new compression method we observe exponential efficiency gains as read lengths increase, and the magnitude of this efficiency gain can be controlled by changing the amount of quality information stored. Our compression method is tunable: The storage of quality scores and unaligned sequences may be adjusted for different experiments to conserve information or to minimize storage costs, and provides one opportunity to address the threat that increasing DNA sequence volumes will overcome our ability to store the sequences.

Beating-heart coronary artery bypass grafting with miniaturized cardiopulmonary bypass results in a more complete revascularization when compared to off-pump grafting.

The technique of miniaturized cardiopulmonary bypass (M-CPB) for beating-heart coronary artery bypass grafting (CABG) is relatively new and has potential advantages when compared to conventional cardiopulmonary bypass (CPB). M-CPB consists of less tubing length and requires less priming volume. The system is phosphorylcholine coated and results in minimal pump-related inflammatory response and organ injury. Finally, this technique combines the advantages of the off-pump CABG (OPCAB) with the better exposure provided by CPB to facilitate complete revascularization. The hypothesis is that CABG with M-CPB has a better outcome in terms of complete coronary revascularization and perioperative results as that compared to off-pump CABG (OPCAB). In a retrospective study, 302 patients underwent beating-heart CABG, 117 (39%) of them with the use of M-CPB and 185 (61%) with OPCAB. After propensity score matching 62 patients in both groups were demographically similar. The most important intra- and early-postoperative parameters were analyzed. Endpoints were hospital mortality and complete revascularization. Hospital mortality was comparable between the groups. The revascularization was significantly more complete in M-CPB patients than in patients in the OPCAB group. Beating-heart CABG with M-CPB is a safe procedure and it provides an optimal operative exposure with significantly more complete coronary revascularization when compared to OPCAB. Beating-heart CABG with the support of a M-CPB is the operation of choice when total coronary revascularization is needed.

Long-term sinus rhythm stability after intraoperative ablation of permanent atrial fibrillation.

INTRODUCTION: Short- and medium-term sinus rhythm (SR) rates after intraoperative radiofrequency ablation to treat permanent atrial fibrillation (AF) are well documented. Is rhythm success stable during a long-term follow-up? METHODS AND RESULTS: A total of 130 patients who had undergone intraoperative radiofrequency cooled-tip endocardial ablation (SICTRA) of permanent AF (mean AF duration 6+/-5 years) concomitant to open heart surgery more than 3 years ago were followed up using electrocardiogram (ECG), Holter-ECG, and echocardiography and compared with 12-month follow-up data. In 55% of patients, only the left atrium and in 45%, both atria were treated using SICTRA. Mitral valve replacement was performed in 21, mitral valve reconstruction in 25, aortic valve replacement in 13, CABG procedures in 51 (including 11 patients with additional mitral valve surgery), and complex procedures in 20 patients. Sixty-nine percent of patients (90/130) were in stable SR after a median period of 48 months, whereas 28% (36/130) were in AF and 3% (4/130) were in atrial flutter. In between the 12-month follow-up and the long-term follow-up, seven patients converted to AF after having documented SR, two patients converted to typical right atrial flutter after being in SR, and two patients from AF to left atrial macroreentry. After left and biatrial SICTRA, SR rates were comparable (73% vs 66%, P = 0.45). Echocardiography revealed 73% of patients in SR to have effective left atrial contraction. CONCLUSIONS: SICTRA restores long-term stable SR in 69% of all patients. Nine percent of patients reconverted back to atrial arrhythmia after having documented SR at 12 months.

Beating-heart coronary artery bypass grafting using a miniaturized extracorporeal circulation system.

BACKGROUND: Experience with miniaturized coronary artery bypass (CAB) systems in coronary artery bypass graft (CABG) surgery on the beating heart is limited. We used a relatively new miniaturized cardiopulmonary bypass (CPB) system, which we termed assisted CAB (ACAB), to perform CABG on the beating heart in 110 patients, and we analyzed clinical outcomes in this patient group. METHODS: Between January 2004 and September 2006, we used ACAB to perform CABG on the beating heart in 110 patients. The mean patient age was 73 +/- 8.1 years. The ACAB system uses a small prime volume of only 500 mL, and the circuit is shorter than that used in conventional CPB. In addition, the tubing and oxygenator systems were surface-coated with phosphorylcholine. The initial heparin dose was 150 IU/kg, with a target activated clotting time of >250 seconds. With this management, none of the patients experienced system thrombosis. We did not use cardioplegia or aortic crossclamping and did not routinely retransfuse cardiotomy blood. Observational data for the 110 patients were analyzed. RESULTS: The mean number of anastomoses performed was 2.67. The rate of perioperative infarction was 1.8% (2 patients). Perioperative mortality was 7% (8 patients). The mean EuroSCORE for all patients was 6.4 +/- 4, whereas it was 13.75 +/- 6.18 for the patients who died. Mean CPB time was 64.96 +/- 16.66 minutes. CONCLUSION: In our experience, beating heart CABG supported by a miniaturized CPB is a safe procedure with acceptable perioperative results.

Clinical outcome in patients with intermediate or equivocal left main coronary artery disease after deferral of surgical revascularization on the basis of fractional flow reserve measurements.

BACKGROUND: Correct assessment of the significance of left main stem lesions is of pivotal importance to the patient with coronary artery disease. On the basis of clinical and angiographic information alone, this evaluation often cannot be done reliably. Limited data suggest that coronary pressure-derived fractional flow reserve (FFR) supports decision making in equivocal left main disease. METHODS: All patients presenting to our institution between June 1999 and June 2004 with intermediate left main coronary artery disease (40%-80% diameter stenosis by angiography), or in whom left main coronary disease was suspected but could not be quantified angiographically, were included in this prospective single-center follow-up study. If FFR was <0.75 along the left main stem, surgical revascularization was recommended; if FFR was >0.80, medical treatment or percutaneous coronary intervention elsewhere in the coronary tree was chosen. If FFR was in the "gray zone," between > or = 0.75 and < or = 0.80 treatment recommendation was dependent on additional individual criteria. Primary end points were freedom from death, myocardial infarction, any coronary revascularization procedure, and stroke. RESULTS: Fifty-one patients (mean age 62.2 +/- 9.6 years, 41 male) were included. In 27 patients (53%), coronary artery bypass surgery was performed. The remaining 24 patients (47%) were treated nonsurgically. Mean follow-up was 29 +/- 16 months. Estimated survival after 4 years of follow-up was 81% among patients in the surgical group and 100% among patients in the nonsurgical group. Event-free survival was 66% in the surgical group and 69% in the nonsurgical group. CONCLUSIONS: Fractional flow reserve is helpful to identify patients with intermediate left main disease in whom deferral of surgical revascularization is associated with excellent survival and low event rates.

Severity of mitral regurgitation may be underestimated in the presence of a left atrial myxoma.

It is well known that mobile or large left atrial myxoma may lead to valve obstruction and insufficiency, and affect transmitral valve flow. The case is reported of a 47-year-old woman with severe mitral regurgitation (MR) and huge left atrial myxoma (6?4?3 cm). Preoperatively, the MR appeared related to the myxoma and was classified as moderate. Following extirpation of the myxoma, intraoperative transesophageal echocardiography (TEE) revealed a severe mitral regurgitant jet. After mitral valve repair by ring implantation, the MR was reduced to minor insufficiency. Hence, a large atrial tumor may mask the severity of concomitant unrelated MR in routine echocardiography. Intraoperative TEE helped in assessing correct mitral valve function after tumor extirpation. Preoperative echocardiography should focus on mitral valve dysfunction in order to separate tumor-related and -unrelated valvular pathologies.

Optimizing revascularization strategies in patients with multivessel coronary disease: impact of intracoronary pressure measurements.

OBJECTIVES: In patients with multivessel coronary disease, the functional significance of each lesion is often unclear, and preinterventional stress tests may be inconclusive. In this setting, intracoronary pressure measurements may be helpful to define the optimal revascularization strategy. METHODS: Twenty-five consecutive patients (aged 64 +/- 11 years) with multivessel disease, inconclusive stress tests or not performed stress tests, and an angiographically intermediate coronary artery stenosis in at least 1 major vessel underwent intracoronary pressure measurements. Myocardial fractional flow reserve was measured for the intermediate lesions under the condition of maximum hyperemia induced by intravenous adenosine (140 microg x kg(-1) x min(-1). Revascularization strategies based on angiographic information alone were compared with treatment strategies based on fractional flow reserve results. RESULTS: The original recommendation of the revascularization procedure of choice (bypass operation or angioplasty) was changed in 9 patients (36%) on the basis of the results of fractional flow reserve measurements. In 6 more patients, pressure measurements led to a change in the recommended number of anastomoses to be aimed for during the operation. Within diffusely diseased vessels, fractional flow reserve provided an exact segmental resolution of pathologic vessel resistance for optimal graft placement. Significant left main disease was confirmed in 3 of 6 patients and was detected in 3 angiographically unsuspected cases. CONCLUSIONS: In patients with multivessel disease, coronary pressure-derived fractional flow reserve is a valuable tool to guide clinical decision making and support cardiologists and cardiovascular surgeons in the composition of optimal revascularization strategies.

Genomic Analysis and Isolation of RNA Polymerase II Dependent Promoters from Spodoptera frugiperda.

The Baculoviral Expression Vector System (BEVS) is the most commonly used method for high expression of recombinant protein in insect cells. Nevertheless, expression of some target proteins--especially those entering the secretory pathway--provides a severe challenge for the baculovirus infected insect cells, due to the reorganisation of intracellular compounds upon viral infection. Therefore, alternative strategies for recombinant protein production in insect cells like transient plasmid-based expression or stable expression cell lines are becoming more popular. However, the major bottleneck of these systems is the lack of strong endogenous polymerase II dependent promoters, as the strong baculoviral p10 and polH promoters used in BEVS are only functional in presence of the viral transcription machinery during the late phase of infection. In this work we present a draft genome and a transcriptome analysis of Sf21 cells for the identification of the first known endogenous Spodoptera frugiperda promoters. Therefore, putative promoter sequences were identified and selected because of high mRNA level or in analogy to other strong promoters in other eukaryotic organism. The chosen endogenous Sf21 promoters were compared to early viral promoters for their efficiency to trigger eGFP expression using transient plasmid based transfection in a BioLector Microfermentation system. Furthermore, promoter activity was not only shown in Sf21 cells but also in Hi5 cells. The novel endogenous Sf21 promoters were ranked according to their activity and expand the small pool of available promoters for stable insect cell line development and transient plasmid expression in insect cells. The best promoter was used to improve plasmid based transient transfection in insect cells substantially.

CBFB-MYH11 hypomethylation signature and PBX3 differential methylation revealed by targeted bisulfite sequencing in patients with acute myeloid leukemia.

BACKGROUND: Studying DNA methylation changes in the context of structural rearrangements and point mutations as well as gene expression changes enables the identification of genes that are important for disease onset and progression in different subtypes of acute myeloid leukemia (AML) patients. The aim of this study was to identify differentially methylated genes with potential impact on AML pathogenesis based on the correlation of methylation and expression data. METHODS: The primary method of studying DNA methylation changes was targeted bisulfite sequencing capturing approximately 84 megabases (Mb) of the genome in 14 diagnostic AML patients and a healthy donors' CD34+ pool. Subsequently, selected DNA methylation changes were confirmed by 454 bisulfite pyrosequencing in a larger cohort of samples. Furthermore, we addressed gene expression by microarray profiling and correlated methylation of regions adjacent to transcription start sites with expression of corresponding genes. RESULTS: Here, we report a novel hypomethylation pattern, specific to CBFB-MYH11 fusion resulting from inv(16) rearrangement that is associated with genes previously described as upregulated in inv(16) AML. We assume that this hypomethylation and corresponding overexpresion occurs in the genes whose function is important in inv(16) leukemogenesis. Further, by comparing all targeted methylation and microarray expression data, PBX3 differential methylation was found to correlate with its gene expression. PBX3 has been recently shown to be a key interaction partner of HOX genes during leukemogenesis and we revealed higher incidence of relapses in PBX3-overexpressing patients. CONCLUSIONS: We discovered new genomic regions with aberrant DNA methylation that are associated with expression of genes involved in leukemogenesis. Our results demonstrate the potential of the targeted approach for DNA methylation studies to reveal new regulatory regions.

Manual repositioning of intra-atrial kidney cancer tumor thrombus: a technique reducing the need for cardiopulmonary bypass.

OBJECTIVE: To describe a feasible surgical technique for patients with renal cell carcinoma associated with a supradiaphragmatic tumor thrombus that avoids cardiopulmonary bypass procedure. MATERIALS AND METHODS: From 2004 to 2009, 4 patients with a right kidney tumor and tumor thrombus above the diaphragm (pT3c) underwent manual repositioning of the tumor thrombus out of the right atrium into the inferior vena cava on the beating heart. These patients were aged 65.8 years and had a body mass index of 25.5 kg/m(2). Median tumor size was 10.8 cm, and 3 patients had synchronous metastasis. RESULTS: Manual repositioning of the tumor thrombus was safe and feasible in all patients. Mean operating time was 561 minutes (range, 302-613 minutes), and no perioperative death occurred. Auxiliary cardiopulmonary bypass procedure was applied in 1 patient to remove a preoperatively diagnosed pulmonary embolus. Three patients subsequently underwent systemic therapy for metastatic disease. Median survival was 16 months (range, 1.7-26 months). CONCLUSION: Manual repositioning of a vena cava tumor thrombus without cardiopulmonary bypass is a safe and feasible approach. The risk of tumor thrombembolization seems to be low, and cardiopulmonary bypass can be avoided or at least reduced to a minimum time of intervention.

A draft sequence of the Neandertal genome.

Neandertals, the closest evolutionary relatives of present-day humans, lived in large parts of Europe and western Asia before disappearing 30,000 years ago. We present a draft sequence of the Neandertal genome composed of more than 4 billion nucleotides from three individuals. Comparisons of the Neandertal genome to the genomes of five present-day humans from different parts of the world identify a number of genomic regions that may have been affected by positive selection in ancestral modern humans, including genes involved in metabolism and in cognitive and skeletal development. We show that Neandertals shared more genetic variants with present-day humans in Eurasia than with present-day humans in sub-Saharan Africa, suggesting that gene flow from Neandertals into the ancestors of non-Africans occurred before the divergence of Eurasian groups from each other.