Ignas Bunikis

Data-driven unbiased curation of the TP53 tumor suppressor gene mutation database and validation by ultradeep sequencing of human tumors

Cancer mutation databases are expected to play central roles in personalized medicine by providing targets for drug development and biomarkers to tailor treatments to each patient. The accuracy of reported mutations is a critical issue that is commonly overlooked, which leads to mutation databases that include a sizable number of spurious mutations, either sequencing errors or passenger mutations. Here we report an analysis of the latest version of the TP53 mutation database, including 34,453 mutations. By using several data-driven methods on multiple independent quality criteria, we obtained a quality score for each report contributing to the database. This score can now be used to filter for high-confidence mutations and reports within the database. Sequencing the entire TP53 gene from various types of cancer using next-generation sequencing with ultradeep coverage validated our approach for curation. In summary, 9.7% of all collected studies, mostly comprising numerous tumors with multiple infrequent TP53 mutations, should be excluded when analyzing TP53 mutations. Thus, by combining statistical and experimental analyses, we provide a curated mutation database for TP53 mutations and a framework for mutation database analysis.

Transcriptome of the Alternative Ethanol Production Strain Dekkera bruxellensis CBS 11270 in Sugar Limited, Low Oxygen Cultivation

Dekkera bruxellensis can outcompete Saccharomyces cerevisiae in environments with low sugar concentrations. It is usually regarded as a spoilage yeast but has lately been identified as an alternative ethanol production organism. In this study, global gene expression in the industrial isolate D. bruxellensis CBS 11270 under oxygen and glucose limitation was investigated by whole transcriptome sequencing using the AB SOLiD technology. Among other observations, we noted expression of respiratory complex I NADH-ubiquinone reductase although D. bruxellensis is a Crabtree positive yeast. The observed higher expression of NADH-generating enzymes compared to NAD+-generating enzymes might be the reason for the previously observed NADH imbalance and resulting Custer effect in D. bruxellensis. Low expression of genes involved in glycerol production is probably the molecular basis for high efficiency of D. bruxellensis metabolism under nutrient limitation. No D. bruxellensis homologs to the genes involved in the final reactions of glycerol biosynthesis were detected. A high number of expressed sugar transporter genes is consistent with the hypothesis that the competitiveness of D. bruxellensis is due to a higher affinity for the limiting substrate.

Combination of short-read, long-read and optical mapping assemblies reveals large-scale tandem repeat arrays with population genetic implications

Accurate and contiguous genome assembly is key to a comprehensive understanding of the processes shaping genomic diversity and evolution. Yet, it is frequently constrained by constitutive heterochromatin, usually characterized by highly repetitive DNA. As a key feature of genome architecture associated with centromeric and subtelomeric regions, it locally influences meiotic recombination. In this study, we assess the impact of large tandem repeat arrays on the recombination rate landscape in an avian speciation model, the Eurasian crow. We assembled two high-quality genome references using single-molecule real-time sequencing (long-read assembly [LR]) and single-molecule optical maps (optical map assembly [OM]). A three-way comparison including the published short-read assembly (SR) constructed for the same individual allowed assessing assembly properties and pinpointing misassemblies. By combining information from all three assemblies, we characterized 36 previously unidentified large repetitive regions in the proximity of sequence assembly breakpoints, the majority of which contained complex arrays of a 14-kb satellite repeat or its 1.2-kb subunit. Using whole-genome population resequencing data, we estimated the population-scaled recombination rate (ρ) and found it to be significantly reduced in these regions. These findings are consistent with an effect of low recombination in regions adjacent to centromeric or subtelomeric heterochromatin and add to our understanding of the processes generating widespread heterogeneity in genetic diversity and differentiation along the genome. By combining three different technologies, our results highlight the importance of adding a layer of information on genome structure that is inaccessible to each approach independently.

Comprehensive profiling of the vaginal microbiome in HIV positive women using massive parallel semiconductor sequencing

Infections by HIV increase the risk of acquiring secondary viral and bacterial infections and methods are needed to determine the spectrum of co-infections for proper treatment. We used rolling circle amplification (RCA) and Ion Proton sequencing to investigate the vaginal microbiome of 20 HIV positive women from South Africa. A total of 46 different human papillomavirus (HPV) types were found, many of which are not detected by existing genotyping assays. Moreover, the complete genomes of two novel HPV types were determined. Abundance of HPV infections was highly correlated with real-time PCR estimates, indicating that the RCA-Proton method can be used for quantification of individual pathogens. We also identified a large number of other viral, bacterial and parasitic co-infections and the spectrum of these co-infections varied widely between individuals. Our method provides rapid detection of a broad range of pathogens and the ability to reconstruct complete genomes of novel infectious agents.

CanvasDB: a local database infrastructure for analysis of targeted- and whole genome re-sequencing projects

CanvasDB is an infrastructure for management and analysis of genetic variants from massively parallel sequencing (MPS) projects. The system stores SNP and indel calls in a local database, designed to handle very large datasets, to allow for rapid analysis using simple commands in R. Functional annotations are included in the system, making it suitable for direct identification of disease-causing mutations in human exome- (WES) or whole-genome sequencing (WGS) projects. The system has a built-in filtering function implemented to simultaneously take into account variant calls from all individual samples. This enables advanced comparative analysis of variant distribution between groups of samples, including detection of candidate causative mutations within family structures and genome-wide association by sequencing. In most cases, these analyses are executed within just a matter of seconds, even when there are several hundreds of samples and millions of variants in the database. We demonstrate the scalability of canvasDB by importing the individual variant calls from all 1092 individuals present in the 1000 Genomes Project into the system, over 4.4 billion SNPs and indels in total. Our results show that canvasDB makes it possible to perform advanced analyses of large-scale WGS projects on a local server. Database URL: https://github.com/UppsalaGenomeCenter/CanvasDB

De novo assembly of Dekkera bruxellensis: a multi technology approach using short and long-read sequencing and optical mapping.

BackgroundIt remains a challenge to perform de novo assembly using next-generation sequencing (NGS). Despite the availability of multiple sequencing technologies and tools (e.g., assemblers) it is still difficult to assemble new genomes at chromosome resolution (i.e., one sequence per chromosome). Obtaining high quality draft assemblies is extremely important in the case of yeast genomes to better characterise major events in their evolutionary history. The aim of this work is two-fold: on the one hand we want to show how combining different and somewhat complementary technologies is key to improving assembly quality and correctness, and on the other hand we present a de novo assembly pipeline we believe to be beneficial to core facility bioinformaticians. To demonstrate both the effectiveness of combining technologies and the simplicity of the pipeline, here we present the results obtained using the Dekkera bruxellensis genome.MethodsIn this work we used short-read Illumina data and long-read PacBio data combined with the extreme long-range information from OpGen optical maps in the task of de novo genome assembly and finishing. Moreover, we developed NouGAT, a semi-automated pipeline for read-preprocessing, de novo assembly and assembly evaluation, which was instrumental for this work.ResultsWe obtained a high quality draft assembly of a yeast genome, resolved on a chromosomal level. Furthermore, this assembly was corrected for mis-assembly errors as demonstrated by resolving a large collapsed repeat and by receiving higher scores by assembly evaluation tools. With the inclusion of PacBio data we were able to fill about 5 % of the optical mapped genome not covered by the Illumina data.

De Novo Assembly of Two Swedish Genomes Reveals Missing Segments from the Human GRCh38 Reference and Improves Variant Calling of Population-Scale Sequencing Data

The current human reference sequence (GRCh38) is a foundation for large-scale sequencing projects. However, recent studies have suggested that GRCh38 may be incomplete and give a suboptimal representation of specific population groups. Here, we performed a de novo assembly of two Swedish genomes that revealed over 10 Mb of sequences absent from the human GRCh38 reference in each individual. Around 6 Mb of these novel sequences (NS) are shared with a Chinese personal genome. The NS are highly repetitive, have an elevated GC-content, and are primarily located in centromeric or telomeric regions. Up to 1 Mb of NS can be assigned to chromosome Y, and large segments are also missing from GRCh38 at chromosomes 14, 17, and 21. Inclusion of NS into the GRCh38 reference radically improves the alignment and variant calling from short-read whole-genome sequencing data at several genomic loci. A re-analysis of a Swedish population-scale sequencing project yields > 75,000 putative novel single nucleotide variants (SNVs) and removes > 10,000 false positive SNV calls per individual, some of which are located in protein coding regions. Our results highlight that the GRCh38 reference is not yet complete and demonstrate that personal genome assemblies from local populations can improve the analysis of short-read whole-genome sequencing data.

A hybrid de novo genome assembly of the honeybee, Apis mellifera, with chromosome-length scaffolds

Background The ability to generate long sequencing reads and access long-range linkage information is revolutionizing the quality and completeness of genome assemblies. Here we use a hybrid approach that combines data from four genome sequencing and mapping technologies to generate a new genome assembly of the honeybee Apis mellifera. We first generated contigs based on PacBio sequencing libraries, which were then merged with linked-read 10x Chromium data followed by scaffolding using a BioNano optical genome map and a Hi-C chromatin interaction map, complemented by a genetic linkage map. Results Each of the assembly steps reduced the number of gaps and incorporated a substantial amount of additional sequence into scaffolds. The new assembly (Amel_HAv3) is significantly more contiguous and complete than the previous one (Amel_4.5), based mainly on Sanger sequencing reads. N50 of contigs is 120-fold higher (5.381 Mbp compared to 0.053 Mbp) and we anchor >98% of the sequence to chromosomes. All of the 16 chromosomes are represented as single scaffolds with an average of three sequence gaps per chromosome. The improvements are largely due to the inclusion of repetitive sequence that was unplaced in previous assemblies. In particular, our assembly is highly contiguous across centromeres and telomeres and includes hundreds of AvaI and AluI repeats associated with these features. Conclusions The improved assembly will be of utility for refining gene models, studying genome function, mapping functional genetic variation, identification of structural variants, and comparative genomics.

Amplicon sequencing of the 16S-ITS-23S rRNA operon with long-read technology for improved phylogenetic classification of uncultured prokaryotes

Amplicon sequencing of the 16S rRNA gene is the predominant method to quantify microbial compositions of environmental samples and to discover previously unknown lineages. Its unique structure of interspersed conserved and variable regions is an excellent target for PCR and allows for classification of reads at all taxonomic levels. However, the relatively few phylogenetically informative sites prevent confident phylogenetic placements of novel lineages that are deep branching relative to reference taxa. This problem is exacerbated when only short 16S rRNA gene fragments are sequenced. To resolve their placement, it is common practice to gather more informative sites by combining multiple conserved genes into concatenated datasets. This however requires genomic data which may be obtained through relatively expensive metagenome sequencing and computationally demanding analyses. Here we develop a protocol that amplifies a large part of 16S and 23S rRNA genes within the rRNA operon, including the ITS region, and sequences the amplicons with PacBio long-read technology. We tested our method with a synthetic mock community and developed a read curation pipeline that reduces the overall error rate to 0.18%. Applying our method on four diverse environmental samples, we were able to capture near full-length rRNA operon amplicons from a large diversity of prokaryotes. Phylogenetic trees constructed with these sequences showed an increase in statistical support compared to trees inferred with shorter, Illumina-like sequences using only the 16S rRNA gene (250 bp). Our method is a cost-effective solution to generate high quality, near full-length 16S and 23S rRNA gene sequences from environmental prokaryotes.

Abstract 2688: A forward genetics screen of murine brain tumors identifies novel candidate genes involved in gliomagenesis

Glioma is the most frequent malignant brain tumor in adults. Platelet-derived growth factor (PDGF) signaling is commonly activated in glioma. We have used a retrovirus-driven PDGFB-induced murine glioma model that causes tumors that closely resemble human gliomas of various grades. Knowing that retroviruses have a capacity to induce insertional mutagenesis, we have employed whole genome sequencing to identify potential genes that, together with PDGFB, drive glioma development. Gliomas were induced by RCAS virus injection into the brains of mice expressing the RCAS retroviral receptor from specific promoters. Genomic DNA from tumor cell lines was probed for retroviral tags and sequenced to identify genomic targets of the retrovirus. A streamlined analysis pipeline was developed for retrovirus integration detection and mapping to the reference mouse genome. Integration sites were analyzed and a common integration site (CIS) label was assigned to a gene, given that it was either tagged by a retrovirus more than once within a discovery set or found within the Retroviral Tagged Cancer Gene Database (RTCGD). In a small discovery subset of 15 murine gliomas, we have identified 40 CIS, of which 37 were validated by Sanger sequencing. When compared with previously identified CIS in RTCGD, 5.5% of them were shared with our older screen, where we overexpressed PDGFB from another retrovirus in order to induce glioma. Less CIS genes were shared with other published tumor models induced by viruses driven by other cancer genes/viruses. The majority of genes identified in our screen were tagged twice. However, Nfic, Cuecd1, Thra, Foxj1 and Nrxn1 were tagged three times, Ppfibp1 and Rhbg four times, and Mir29a/29b-1 seven times. As compared to control tumor lines, two top candidate genes, Mir29a and Ppfibp1, demonstrated significantly increased expression in tumor lines in were they were respectively tagged. Mir29a is often found downregulated in human tumors including gliomas, still high levels of Mir29a are sometimes found in certain aggressive cancers and in metastases. Interestingly, we found that specific PDGFR inhibition negatively regulates Mir29a, indicating a possible role for PDGF signaling in Mir29a regulation. Ppfibp1 has not been extensively studied in cancer. However, Ppfibp1 seems to have a subgroup-specific expression in human glioblastoma, making it an interesting candidate for further analysis. Here we present a new screening method that can be employed to identify genes involved in PDGFB-driven gliomagenesis. So far, we have identified 37 candidate genes by whole genome sequencing. Two of the most frequently tagged candidates, Mir29a and Ppfibp1 were upregulated as a consequence of retroviral mutagenesis. Their precise role in driving glioma formation in collaboration with PDGF is currently explored. Citation Format: Matko Cancer, Holger Weishaupt, Gabriela Rosen, Ignas Bunikis, Yiwen Jiang, Smitha Sreedharan, Sara Bolin, Ulf Gyllensten, Oren J. Becher, Lene Uhrbom, Adam Ameur, Fredrik J. Swartling. A forward genetics screen of murine brain tumors identifies novel candidate genes involved in gliomagenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2688.