ITIS,a bioinformatics tool for accurate identification of transposon insertion sites using next-generation sequencing data

Background

Transposable elements constitute an important part of the genome and are essential in adaptive mechanisms. Transposition events associated with phenotypic changes occur naturally or are induced in insertional mutant populations. Transposon mutagenesis results in multiple random insertions and recovery of most/all the insertions is critical for forward genetics study. Using genome next-generation sequencing data and appropriate bioinformatics tool, it is plausible to accurately identify transposon insertion sites, which could provide candidate causal mutations for desired phenotypes for further functional validation.

Results

We developed a novel bioinformatics tool, ITIS (Identification of Transposon Insertion Sites), for localizing transposon insertion sites within a genome. It takes next-generation genome re-sequencing data (NGS data), transposon sequence, and reference genome sequence as input, and generates a list of highly reliable candidate insertion sites as well as zygosity information of each insertion. Using a simulated dataset and a case study based on an insertional mutant line from Medicago truncatula, we showed that ITIS performed better in terms of sensitivity and specificity than other similar algorithms such as RelocaTE, RetroSeq, TEMP and TIF. With the case study data, we demonstrated the efficiency of ITIS by validating the presence and zygosity of predicted insertion sites of the Tnt1 transposon within a complex plant system, M. truncatula.

Conclusion

This study showed that ITIS is a robust and powerful tool for forward genetic studies in identifying transposable element insertions causing phenotypes. ITIS is suitable in various systems such as cell culture, bacteria, yeast, insect, mammal and plant.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-015-0507-2) contains supplementary material, which is available to authorized users.     

VISA - Vector Integration Site Analysis server: a web-based server to rapidly identify retroviral integration sites from next-generation sequencing

Background

Analyzing the integration profile of retroviral vectors is a vital step in determining their potential genotoxic effects and developing safer vectors for therapeutic use. Identifying retroviral vector integration sites is also important for retroviral mutagenesis screens.

Results

We developed VISA, a vector integration site analysis server, to analyze next-generation sequencing data for retroviral vector integration sites. Sequence reads that contain a provirus are mapped to the human genome, sequence reads that cannot be localized to a unique location in the genome are filtered out, and then unique retroviral vector integration sites are determined based on the alignment scores of the remaining sequence reads.

Conclusions

VISA offers a simple web interface to upload sequence files and results are returned in a concise tabular format to allow rapid analysis of retroviral vector integration sites.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-015-0653-6) contains supplementary material, which is available to authorized users.     

HotSpotter: efficient visualization of driver mutations

Background

Driver mutations are positively selected during the evolution of cancers. The relative frequency of a particular mutation within a gene is typically used as a criterion for identifying a driver mutation. However, driver mutations may occur with relative infrequency at a particular site, but cluster within a region of the gene. When analyzing across different cancers, particular mutation sites or mutations within a particular region of the gene may be of relatively low frequency in some cancers, but still provide selective growth advantage.

Results

This paper presents a method that allows rapid and easy visualization of mutation data sets and identification of potential gene mutation hotspot sites and/or regions. As an example, we identified hotspot regions in the NFE2L2 gene that are potentially functionally relevant in endometrial cancer, but would be missed using other analyses.

Conclusions

HotSpotter is a quick, easy-to-use visualization tool that delivers gene identities with associated mutation locations and frequencies overlaid upon a large cancer mutation reference set. This allows the user to identify potential driver mutations that are less frequent in a cancer or are localized in a hotspot region of relatively infrequent mutations.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1044) contains supplementary material, which is available to authorized users.     

Discovery of transgene insertion sites by high throughput sequencing of mate pair libraries

Background

Transgenesis by random integration of a transgene into the genome of a zygote has become a reliable and powerful method for the creation of new mouse strains that express exogenous genes, including human disease genes, tissue specific reporter genes or genes that allow for tissue specific recombination. Nearly 6,500 transgenic alleles have been created by random integration in embryos over the last 30 years, but for the vast majority of these strains, the transgene insertion sites remain uncharacterized.

Results

To obtain a complete understanding of how insertion sites might contribute to phenotypic outcomes, to more cost effectively manage transgenic strains, and to fully understand mechanisms of instability in transgene expression, we’ve developed methodology and a scoring scheme for transgene insertion site discovery using high throughput sequencing data.

Conclusions

Similar to other molecular approaches to transgene insertion site discovery, high-throughput sequencing of standard paired-end libraries is hindered by low signal to noise ratios. This problem is exacerbated when the transgene consists of sequences that are also present in the host genome. We’ve found that high throughput sequencing data from mate-pair libraries are more informative when compared to data from standard paired end libraries. We also show examples of the genomic regions that harbor transgenes, which have in common a preponderance of repetitive sequences.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-367) contains supplementary material, which is available to authorized users.     

The distribution and mutagenesis of short coding INDELs from 1,128 whole exomes

Background

Identifying insertion/deletion polymorphisms (INDELs) with high confidence has been intrinsically challenging in short-read sequencing data. Here we report our approach for improving INDEL calling accuracy by using a machine learning algorithm to combine call sets generated with three independent methods, and by leveraging the strengths of each individual pipeline. Utilizing this approach, we generated a consensus exome INDEL call set from a large dataset generated by the 1000 Genomes Project (1000G), maximizing both the sensitivity and the specificity of the calls.

Results

This consensus exome INDEL call set features 7,210 INDELs, from 1,128 individuals across 13 populations included in the 1000 Genomes Phase 1 dataset, with a false discovery rate (FDR) of about 7.0%.

Conclusions

In our study we further characterize the patterns and distributions of these exonic INDELs with respect to density, allele length, and site frequency spectrum, as well as the potential mutagenic mechanisms of coding INDELs in humans.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1333-7) contains supplementary material, which is available to authorized users.     

Generation of a predictive melphalan resistance index by drug screen of B-cell cancer cell lines

Background

Recent reports indicate that in vitro drug screens combined with gene expression profiles (GEP) of cancer cell lines may generate informative signatures predicting the clinical outcome of chemotherapy. In multiple myeloma (MM) a range of new drugs have been introduced and now challenge conventional therapy including high dose melphalan. Consequently, the generation of predictive signatures for response to melphalan may have a clinical impact. The hypothesis is that melphalan screens and GEPs of B-cell cancer cell lines combined with multivariate statistics may provide predictive clinical information.

Materials and Methods

Microarray based GEPs and a melphalan growth inhibition screen of 59 cancer cell lines were downloaded from the National Cancer Institute database. Equivalent data were generated for 18 B-cell cancer cell lines. Linear discriminant analyses (LDA), sparse partial least squares (SPLS) and pairwise comparisons of cell line data were used to build resistance signatures from both cell line panels. A melphalan resistance index was defined and estimated for each MM patient in a publicly available clinical data set and evaluated retrospectively by Cox proportional hazards and Kaplan-Meier survival analysis.

Principal Findings

Both cell line panels performed well with respect to internal validation of the SPLS approach but only the B-cell panel was able to predict a significantly higher risk of relapse and death with increasing resistance index in the clinical data sets. The most sensitive and resistant cell lines, MOLP-2 and RPMI-8226 LR5, respectively, had high leverage, which suggests their differentially expressed genes to possess important predictive value.

Conclusion

The present study presents a melphalan resistance index generated by analysis of a B-cell panel of cancer cell lines. However, the resistance index needs to be functionally validated and correlated to known MM biomarkers in independent data sets in order to better understand the mechanism underlying the preparedness to melphalan resistance.     

The Specificity of the FOXL2 c.402C>G Somatic Mutation: A Survey of Solid Tumors

Background

A somatic mutation in the FOXL2 gene is reported to be present in almost all (97%; 86/89) morphologically defined, adult-type, granulosa-cell tumors (A-GCTs). This FOXL2 c.402C>G mutation changes a highly conserved cysteine residue to a tryptophan (p.C134W). It was also found in a minority of other ovarian malignant stromal tumors, but not in benign ovarian stromal tumors or unrelated ovarian tumors or breast cancers.

Methodology/Principal Findings

Herein we studied other cancers and cell lines for the presence of this mutation. We screened DNA from 752 tumors of epithelial and mesenchymal origin and 28 ovarian cancer cell lines and 52 other cancer cell lines of varied origin. We found the FOXL2 c.402C>G mutation in an unreported A-GCT case and the A-GCT-derived cell line KGN. All other tumors and cell lines analyzed were mutation negative.

Conclusions/Significance

In addition to proving that the KGN cell line is a useful model to study A-GCTs, these data show that the c.402C>G mutation in FOXL2 is not commonly found in a wide variety of other cancers and therefore it is likely pathognomonic for A-GCTs and closely related tumors.     

A Randomised Trial Evaluating the Effects of the TRPV1 Antagonist SB705498 on Pruritus Induced by Histamine,and Cowhage Challenge in Healthy Volunteers

Background

Transient receptor potential vanilloid type 1 (TRPV1) is a non-selective cation channel widely expressed in skin tissues, and peripheral sensory nerve fibres. Activation of TRPV1 releases neuropeptides; the resulting neurogenic inflammation is believed to contribute to the development of pruritus. A TRPV1 antagonist has the potential to perform as an anti-pruritic agent. SB705498 is a TRPV1 antagonist that has demonstrated in vitro activity against cloned TRPV1 human receptors and when orally administered has demonstrated pharmacodynamic activity in animal models and clinical studies.

Objectives

To select a topical dose of SB705498 using the TRPV1 agonist capsaicin; to confirm engagement of the TRPV1 antagonistic action of SB705498 and assess whether the dose selected has an effect on itch induced by two challenge agents.

Methods

A clinical study was conducted in 16 healthy volunteers to assess the effects of 3 doses of SB705498 on skin flare induced by capsaicin. Subjects with a robust capsaicin response were chosen to determine if the selected topical formulation of SB705498 had an effect on challenge agent induced itch.

Results

Following capsaicin challenge the greatest average reduction in area of flare was seen for the 3% formulation. This dose was selected for further investigation. Itch intensity induced by two challenge agents (cowhage and histamine) was assessed on the Computerised Visual Analogue Scale. The difference in average itch intensity (Weighted Mean Over 15 Mins) between the 3% dose of SB705498 and placebo for the cowhage challenge was −0.64, whilst the histamine challenge showed on average a −4.65 point change.

Conclusions

The 3% topical formulation of SB705498 cream was clinically well tolerated and had target specific pharmacodynamic activity. However there were no clinically significant differences on pruritus induced by either challenge agent in comparison to placebo. SB705498 is unlikely to be of symptomatic benefit for histaminergic or non-histaminergic induced itch.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01673529","term_id":"NCT01673529"}}NCT01673529     

Transcervical Ultrasonography Is Feasible to Visualize and Evaluate Base of Tongue Cancers

Background

Base of tongue (BOT) is a difficult subsite to examine clinically and radiographically. Yet, anatomic delineation of the primary tumor site, its extension to adjacent sites or across midline, and endophytic vs. exophytic extent are important characteristics for staging and treatment planning. We hypothesized that ultrasound could be used to visualize and describe BOT tumors.

Methods

Transcervical ultrasound was performed using a standardized protocol in cases and controls. Cases had suspected or confirmed BOT malignancy. Controls were healthy individuals without known malignancy.

Results

100% of BOT tumors were visualized. On ultrasound BOT tumors were hypoechoic (90.9%) with irregular margins (95.5%). Ultrasound could be used to characterize adjacent site involvement, midline extent, and endophytic extent, and visualize the lingual artery. No tumors were suspected for controls.

Conclusions

Ultrasonography can be used to transcervically visualize BOT tumors and provides clinically relevant characteristics that may not otherwise be appreciable.     

Deep sequencing of multiple regions of glial tumors reveals spatial heterogeneity for mutations in clinically relevant genes

Background

The extent of intratumoral mutational heterogeneity remains unclear in gliomas, the most common primary brain tumors, especially with respect to point mutation. To address this, we applied single molecule molecular inversion probes targeting 33 cancer genes to assay both point mutations and gene amplifications within spatially distinct regions of 14 glial tumors.

Results

We find evidence of regional mutational heterogeneity in multiple tumors, including mutations in TP53 and RB1 in an anaplastic oligodendroglioma and amplifications in PDGFRA and KIT in two glioblastomas (GBMs). Immunohistochemistry confirms heterogeneity of TP53 mutation and PDGFRA amplification. In all, 3 out of 14 glial tumors surveyed have evidence for heterogeneity for clinically relevant mutations.

Conclusions

Our results underscore the need to sample multiple regions in GBM and other glial tumors when devising personalized treatments based on genomic information, and furthermore demonstrate the importance of measuring both point mutation and copy number alteration while investigating genetic heterogeneity within cancer samples.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0530-z) contains supplementary material, which is available to authorized users.     

Involvement of a citrus meiotic recombination TTC-repeat motif in the formation of gross deletions generated by ionizing radiation and MULE activation

Background

Transposable-element mediated chromosomal rearrangements require the involvement of two transposons and two double-strand breaks (DSB) located in close proximity. In radiobiology, DSB proximity is also a major factor contributing to rearrangements. However, the whole issue of DSB proximity remains virtually unexplored.

Results

Based on DNA sequencing analysis we show that the genomes of 2 derived mutations, Arrufatina (sport) and Nero (irradiation), share a similar 2 Mb deletion of chromosome 3. A 7 kb Mutator-like element found in Clemenules was present in Arrufatina in inverted orientation flanking the 5′ end of the deletion. The Arrufatina Mule displayed “dissimilar” 9-bp target site duplications separated by 2 Mb. Fine-scale single nucleotide variant analyses of the deleted fragments identified a TTC-repeat sequence motif located in the center of the deletion responsible of a meiotic crossover detected in the citrus reference genome.

Conclusions

Taken together, this information is compatible with the proposal that in both mutants, the TTC-repeat motif formed a triplex DNA structure generating a loop that brought in close proximity the originally distinct reactive ends. In Arrufatina, the loop brought the Mule ends nearby the 2 distinct insertion target sites and the inverted insertion of the transposable element between these target sites provoked the release of the in-between fragment. This proposal requires the involvement of a unique transposon and sheds light on the unresolved question of how two distinct sites become located in close proximity. These observations confer a crucial role to the TTC-repeats in fundamental plant processes as meiotic recombination and chromosomal rearrangements.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1280-3) contains supplementary material, which is available to authorized users.     

Development of synchronous VHL syndrome tumors reveals contingencies and constraints to tumor evolution

Background

Genomic analysis of multi-focal renal cell carcinomas from an individual with a germline VHL mutation offers a unique opportunity to study tumor evolution.

Results

We perform whole exome sequencing on four clear cell renal cell carcinomas removed from both kidneys of a patient with a germline VHL mutation. We report that tumors arising in this context are clonally independent and harbour distinct secondary events exemplified by loss of chromosome 3p, despite an identical genetic background and tissue microenvironment. We propose that divergent mutational and copy number anomalies are contingent upon the nature of 3p loss of heterozygosity occurring early in tumorigenesis. However, despite distinct 3p events, genomic, proteomic and immunohistochemical analyses reveal evidence for convergence upon the PI3K-AKT-mTOR signaling pathway. Four germline tumors in this young patient, and in a second, older patient with VHL syndrome demonstrate minimal intra-tumor heterogeneity and mutational burden, and evaluable tumors appear to follow a linear evolutionary route, compared to tumors from patients with sporadic clear cell renal cell carcinoma.

Conclusions

In tumors developing from a germline VHL mutation, the evolutionary principles of contingency and convergence in tumor development are complementary. In this small set of patients with early stage VHL-associated tumors, there is reduced mutation burden and limited evidence of intra-tumor heterogeneity.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0433-z) contains supplementary material, which is available to authorized users.     

Colorectal Tumors from APC*I1307K Carriers Principally Harbor Somatic APC Mutations outside the A8 Tract

Purpose

APC*I1307K (c.3920T>A) is an inherited variant associated with colorectal tumour risk found almost exclusively in those of Ashkenazi Jewish ancestry. A single nucleotide substitution creates an oligo-adenine tract (A8) that appears to be inherently prone to further mis-pairing and slippage. The reported multiple tumor phenotype of carriers is not easily reconciled with molecular and population genetics data. We postulated that some c.3920T>A carriers with multiple adenomas have other unidentified APC germ line or somatic mutations.

Methods

DNA from 82 colonic tumours and accompanying normal tissue collected from 29 carriers with multiple colorectal tumors was directly sequenced between codons 716 and 1604. We also assessed APC gene loss of heterozygosity.

Results

One patient (3.4%) was found to have an additional APC germ line mutation. Twenty-five of the tumours showed no significant somatic molecular change, 36 showed one change, 20 showed two, and one tumour showed more than 2 changes. Our data suggest a correlation between advancing histology and fewer beta-catenin binding sites remaining in the mutant proteins.

Conclusions

There were no other common germ line variants identified within the region of the APC gene examined, suggesting that any effect from this region on tumour production is attributable to the c.3920T>A allele. Our findings further suggest the only somatic genetic change clearly attributable to the c.3920T>A mutation is the c.3924_3925insA.     

Late foliar diseases in wheat crops decrease nitrogen yield through N uptake rather than through variations in N remobilization

Background and Aims

French wheat grains may be of little value on world markets because they have low and highly variable grain protein concentrations (GPC). This nitrogen-yield to yield ratio depends on crop nitrogen (N) fertilization as well as on crop capacity to use N, which is known to vary with climate and disease severity. Here an examination is made of the respective roles that N remobilization and post-anthesis N uptake play in N yield variations; in particular, when wheat crops (Triticum aestivum) are affected by leaf rust (Puccinia triticina) and Septoria tritici blotch (teleomorph Mycosphaerella graminicola).

Methods

Data from a 4-year field experiment was used to analyse N yield variations in wheat crops grown either with a third or no late N fertilization. Natural aerial epidemics ensured a range of disease severity, and fungicide ensured disease-free control plots. The data set of Gooding et al. (2005, Journal of Agricultural Science 143: 503–518) was incorporated in order to enlarge the range of conditions.

Key Results

Post-anthesis N uptake accounted for a third of N yield whilst N remobilization accounted for two-thirds in all crops whether affected by diseases or not. However, variations in N yield were highly correlated with post-anthesis N uptake, more than with N remobilization, in diseased and also healthy crops. Furthermore, N remobilization did not significantly correlate with N yield in healthy crops. These findings matched data from studies using various wheat genotypes under various management and climatic conditions. Leaf area duration (LAD) accurately predicted N remobilization whether or not crops were diseased; in diseased crops, LAD also accurately predicted N uptake.

Conclusions

Under the experimental conditions, N yield variations were closely associated with post-anthesis N uptake in diseased but also in healthy crops. Understanding the respective roles of N uptake and N remobilization in the case of diseased and healthy crops holds the promise of better modelling of variations in N yield, and thus in GPC.Key words: Triticum aestivum, Puccinia triticina, leaf rust, Mycosphaerella graminicola, Septoria tritici blotch, N uptake, N remobilization, N yield, Leaf area duration     

Upper and Lower Urinary Tract Outcomes in Adult Myelomeningocele Patients: A Systematic Review

Background

The introduction of sophisticated treatment of bladder dysfunction and hydrocephalus allows the majority of SB patients to survive into adulthood. However, no systematic review on urological outcome in adult SB patients is available and no follow-up schemes exist.

Objectives

To systematically summarize the evidence on outcome of urinary tract functioning in adult SB patients.

Methods

A literature search in PubMed and Embase databases was done. Only papers published in the last 25 years describing patients with open SB with a mean age >18 years were included. We focused on finding differences in the treatment strategies, e.g., clean intermittent catheterization and antimuscarinic drugs versus early urinary diversion, with regard to long-term renal and bladder outcomes.

Results

A total of 13 articles and 5 meeting abstracts on urinary tract status of adult SB patients were found describing a total of 1564 patients with a mean age of 26.1 years (range 3–74 years, with a few patients <18 years). All were retrospective cohort studies with relatively small and heterogeneous samples with inconsistent reporting of outcome; this precluded the pooling of data and meta-analysis. Total continence was achieved in 449/1192 (37.7%; range 8–85%) patients. Neurological level of the lesion and hydrocephalus were associated with incontinence. Renal function was studied in 1128 adult patients. In 290/1128 (25.7%; range 3–81.8%) patients some degree of renal damage was found and end-stage renal disease was seen in 12/958 (1.3%) patients. Detrusor-sphincter dyssynergy and detrusor-overactivity acted as adverse prognostic factors for the development of renal damage.

Conclusions

These findings should outline follow-up schedules for SB patients, which do not yet exist. Since renal and bladder deterioration continues beyond adolescence, follow-up of these individuals is needed. We recommend standardization in reporting the outcome of urinary tract function in adult SB patients.     

From microbial gene essentiality to novel antimicrobial drug targets

Background

Bacterial respiratory tract infections, mainly caused by Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis are among the leading causes of global mortality and morbidity. Increased resistance of these pathogens to existing antibiotics necessitates the search for novel targets to develop potent antimicrobials.

Result

Here, we report a proof of concept study for the reliable identification of potential drug targets in these human respiratory pathogens by combining high-density transposon mutagenesis, high-throughput sequencing, and integrative genomics. Approximately 20% of all genes in these three species were essential for growth and viability, including 128 essential and conserved genes, part of 47 metabolic pathways. By comparing these essential genes to the human genome, and a database of genes from commensal human gut microbiota, we identified and excluded potential drug targets in respiratory tract pathogens that will have off-target effects in the host, or disrupt the natural host microbiota. We propose 249 potential drug targets, 67 of which are targets for 75 FDA-approved antimicrobials and 35 other researched small molecule inhibitors. Two out of four selected novel targets were experimentally validated, proofing the concept.

Conclusion

Here we have pioneered an attempt in systematically combining the power of high-density transposon mutagenesis, high-throughput sequencing, and integrative genomics to discover potential drug targets at genome-scale. By circumventing the time-consuming and expensive laboratory screens traditionally used to select potential drug targets, our approach provides an attractive alternative that could accelerate the much needed discovery of novel antimicrobials.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-958) contains supplementary material, which is available to authorized users.     

Associations of Physical Activity and Sedentary Behavior with Academic Skills – A Follow-Up Study among Primary School Children

Background

There are no prospective studies that would have compared the relationships of different types of physical activity (PA) and sedentary behavior (SB) with academic skills among children. We therefore investigated the associations of different types of PA and SB with reading and arithmetic skills in a follow-up study among children.

Methods

The participants were 186 children (107 boys, 79 girls, 6–8 yr) who were followed-up in Grades 1–3. PA and SB were assessed using a questionnaire in Grade 1. Reading fluency, reading comprehension and arithmetic skills were assessed using standardized tests at the end of Grades 1–3.

Results

Among all children more recess PA and more time spent in SB related to academic skills were associated with a better reading fluency across Grades 1–3. In boys, higher levels of total PA, physically active school transportation and more time spent in SB related to academic skills were associated with a better reading fluency across the Grades 1–3. Among girls, higher levels of total PA were related to worse arithmetic skills across Grades 1–3. Moreover, total PA was directly associated with reading fluency and arithmetic skills in Grades 1–3 among girls whose parents had a university degree, whereas these relationships were inverse in girls of less educated parents.

Conclusions

Total PA, physically active school transportation and SB related to academic skills may be beneficial for the development of reading skills in boys, whereas factors that are independent of PA or SB may be more important for academic skills in girls.

Trial Registration

ClinicalTrials.gov: {"type":"clinical-trial","attrs":{"text":"NCT01803776","term_id":"NCT01803776"}}NCT01803776