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Abstract Background The majority of published gene-expression studies have used RNA isolated from whole cells, overlooking the potential impact of including nuclear transcriptome in the analyses. In this study, mRNA fractions from the cytoplasm and from whole cells (total RNA) were prepared from three human cell lines and sequenced using massive parallel sequencing. Results For all three cell lines, of about 15000 detected genes approximately 400 to 1400 genes were detected in different amounts in the cytoplasmic and total RNA fractions. Transcripts detected at higher levels in the total RNA fraction had longer coding sequences and higher number of miRNA target sites. Transcripts detected at higher levels in the cytoplasmic fraction were shorter or contained shorter untranslated regions. Nuclear retention of transcripts and mRNA degradation via miRNA pathway might contribute to this differential detection of genes. The consequence of the differential detection was further investigated by comparison to proteomics data. Interestingly, the expression profiles of cytoplasmic and total RNA correlated equally well with protein abundance levels indicating regulation at a higher level. Conclusions We conclude that expression levels derived from the total RNA fraction be regarded as an appropriate estimate of the amount of mRNAs present in a given cell population, independent of the coding sequence length or UTRs.

Abstract Background There is an imperative need for SNP genotyping technologies that are cost-effective per sample with retained high accuracy, throughput and flexibility. We have developed a microarray-based technique and compared it to Pyrosequencing. In the protease-mediated allele-specific extension (PrASE), the protease constrains the elongation reaction and thus prevents incorrect nucleotide incorporation to mismatched 3'-termini primers. Results The assay is automated for 48 genotyping reactions in parallel followed by a tag-microarray detection system. A script automatically visualizes the results in cluster diagrams and assigns the genotypes. Ten polymorphic positions suggested as prothrombotic genetic variations were analyzed with Pyrosequencing and PrASE technologies in 442 samples and 99.8 % concordance was achieved. In addition to accuracy, the robustness and reproducibility of the technique has been investigated. Conclusion The results of this study strongly indicate that the PrASE technology can offer significant improvements in terms of accuracy and robustness and thereof increased number of typeable SNPs.

Objective: This study sought to determine whether 18 F-fluorodeoxyglucose-positron emission tomography/computed tomography could be applied to a murine model of advanced atherosclerotic plaque vulnerability to detect response to therapeutic intervention and changes in lesion stability. Approach and Results: To analyze plaques susceptible to rupture, we fed ApoE -/- mice a high-fat diet and induced vulnerable lesions by cast placement over the carotid artery. After 9 weeks of treatment with orthogonal therapeutic agents (including lipid-lowering and proefferocytic therapies), we assessed vascular inflammation and several features of plaque vulnerability by 18 F-fluorodeoxyglucose-positron emission tomography/computed tomography and histopathology, respectively. We observed that 18 F-fluorodeoxyglucose-positron emission tomography/computed tomography had the capacity to resolve histopathologically proven changes in plaque stability after treatment. Moreover, mean target-to-background ratios correlated with multiple characteristics of lesion instability, including the corrected vulnerability index., Conclusions: These results suggest that the application of noninvasive 18 F-fluorodeoxyglucose-positron emission tomography/computed tomography to a murine model can allow for the identification of vulnerable atherosclerotic plaques and their response to therapeutic intervention. This approach may prove useful as a drug discovery and prioritization method.

Whole-genome sequencing (WGS) is a fundamental technology for research to advance precision medicine, but the limited availability of portable and user-friendly workflows for WGS analyses poses a major challenge for many research groups and hampers scientific progress. Here we present Sarek, an open-source workflow to detect germline variants and somatic mutations based on sequencing data from WGS, whole-exome sequencing (WES), or gene panels. Sarek features (i) easy installation, (ii) robust portability across different computer environments, (iii) comprehensive documentation, (iv) transparent and easy-to-read code, and (v) extensive quality metrics reporting. Sarek is implemented in the Nextflow workflow language and supports both Docker and Singularity containers as well as Conda environments, making it ideal for easy deployment on any POSIX-compatible computers and cloud compute environments. Sarek follows the GATK best-practice recommendations for read alignment and pre-processing, and includes a wide range of software for the identification and annotation of germline and somatic single-nucleotide variants, insertion and deletion variants, structural variants, tumour sample purity, and variations in ploidy and copy number. Sarek offers easy, efficient, and reproducible WGS analyses, and can readily be used both as a production workflow at sequencing facilities and as a powerful stand-alone tool for individual research groups. The Sarek source code, documentation and installation instructions are freely available at https://github.com/nf-core/sarek and at https://nf-co.re/sarek/., Competing Interests: No competing interests were disclosed., (Copyright: © 2020 Garcia M et al.)

The future of human genomics is one that seeks to resolve the entirety of genetic variation through sequencing. The prospect of utilizing genomics for medical purposes require cost-efficient and accurate base calling, long-range haplotyping capability, and reliable calling of structural variants. Short-read sequencing has lead the development towards such a future but has struggled to meet the latter two of these needs. To address this limitation, we developed a technology that preserves the molecular origin of short sequencing reads, with an insignificant increase to sequencing costs. We demonstrate a novel library preparation method for high throughput barcoding of short reads where millions of random barcodes can be used to reconstruct megabase-scale phase blocks.

Here, we present the genome of the industrial ethanol production strain Brettanomyces bruxellensis CBS 11270. The nuclear genome was found to be diploid, containing four chromosomes with sizes of ranging from 2.2 to 4.0 Mbp. A 75 Kbp mitochondrial genome was also identified. Comparing the homologous chromosomes, we detected that 0.32% of nucleotides were polymorphic, i.e. formed single nucleotide polymorphisms (SNPs), 40.6% of them were found in coding regions (i.e. 0.13% of all nucleotides formed SNPs and were in coding regions). In addition, 8,538 indels were found. The total number of protein coding genes was 4897, of them, 4,284 were annotated on chromosomes; and the mitochondrial genome contained 18 protein coding genes. Additionally, 595 genes, which were annotated, were on contigs not associated with chromosomes. A number of genes was duplicated, most of them as tandem repeats, including a six-gene cluster located on chromosome 3. There were also examples of interchromosomal gene duplications, including a duplication of a six-gene cluster, which was found on both chromosomes 1 and 4. Gene copy number analysis suggested loss of heterozygosity for 372 genes. This may reflect adaptation to relatively harsh but constant conditions of continuous fermentation. Analysis of gene topology showed that most of these losses occurred in clusters of more than one gene, the largest cluster comprising 33 genes. Comparative analysis against the wine isolate CBS 2499 revealed 88,534 SNPs and 8,133 indels. Moreover, when the scaffolds of the CBS 2499 genome assembly were aligned against the chromosomes of CBS 11270, many of them aligned completely, some have chunks aligned to different chromosomes, and some were in fact rearranged. Our findings indicate a highly dynamic genome within the species B. bruxellensis and a tendency towards reduction of gene number in long-term continuous cultivation., Competing Interests: The authors have declared that no competing interests exist.

Complex chromosomal rearrangements (CCRs) are rearrangements involving more than two chromosomes or more than two breakpoints. Whole genome sequencing (WGS) allows for outstanding high resolution characterization on the nucleotide level in unique sequences of such rearrangements, but problems remain for mapping breakpoints in repetitive regions of the genome, which are known to be prone to rearrangements. Hence, multiple complementary WGS experiments are sometimes needed to solve the structures of CCRs. We have studied three individuals with CCRs: Case 1 and Case 2 presented with de novo karyotypically balanced, complex interchromosomal rearrangements (46,XX,t(2;8;15)(q35;q24.1;q22) and 46,XY,t(1;10;5)(q32;p12;q31)), and Case 3 presented with a de novo, extremely complex intrachromosomal rearrangement on chromosome 1. Molecular cytogenetic investigation revealed cryptic deletions in the breakpoints of chromosome 2 and 8 in Case 1, and on chromosome 10 in Case 2, explaining their clinical symptoms. In Case 3, 26 breakpoints were identified using WGS, disrupting five known disease genes. All rearrangements were subsequently analyzed using optical maps, linked-read WGS, and short-read WGS. In conclusion, we present a case series of three unique de novo CCRs where we by combining the results from the different technologies fully solved the structure of each rearrangement. The power in combining short-read WGS with long-molecule sequencing or optical mapping in these unique de novo CCRs in a clinical setting is demonstrated., Competing Interests: The authors have declared that no competing interests exist.

Clustered copy number variants (CNVs) as detected by chromosomal microarray analysis (CMA) are often reported as germline chromothripsis. However, such cases might need further investigations by massive parallel whole genome sequencing (WGS) in order to accurately define the underlying complex rearrangement, predict the occurrence mechanisms and identify additional complexities. Here, we utilized WGS to delineate the rearrangement structure of 21 clustered CNV carriers first investigated by CMA and identified a total of 83 breakpoint junctions (BPJs). The rearrangements were further sub-classified depending on the patterns observed: I) Cases with only deletions (n = 8) often had additional structural rearrangements, such as insertions and inversions typical to chromothripsis; II) cases with only duplications (n = 7) or III) combinations of deletions and duplications (n = 6) demonstrated mostly interspersed duplications and BPJs enriched with microhomology. In two cases the rearrangement mutational signatures indicated both a breakage-fusion-bridge cycle process and haltered formation of a ring chromosome. Finally, we observed two cases with Alu- and LINE-mediated rearrangements as well as two unrelated individuals with seemingly identical clustered CNVs on 2p25.3, possibly a rare European founder rearrangement. In conclusion, through detailed characterization of the derivative chromosomes we show that multiple mechanisms are likely involved in the formation of clustered CNVs and add further evidence for chromoanagenesis mechanisms in both "simple" and highly complex chromosomal rearrangements. Finally, WGS characterization adds positional information, important for a correct clinical interpretation and deciphering mechanisms involved in the formation of these rearrangements., Competing Interests: The authors have declared that no competing interests exist.

Urban sewer systems consist of wastewater and stormwater sewers, of which only wastewater is processed before being discharged. Occasionally, misconnections or damages in the network occur, resulting in untreated wastewater entering natural water bodies via the stormwater system. Cultivation of faecal indicator bacteria (e.g. Escherichia coli; E. coli) is the current standard for tracing wastewater contamination. This method is cheap but has limited specificity and mobility. Here, we compared the E. coli culturing approach with two sequencing-based methodologies (Illumina MiSeq 16S rRNA gene amplicon sequencing and Oxford Nanopore MinION shotgun metagenomic sequencing), analysing 73 stormwater samples collected in Stockholm. High correlations were obtained between E. coli culturing counts and frequencies of human gut microbiome amplicon sequences, indicating E. coli is indeed a good indicator of faecal contamination. However, the amplicon data further holds information on contamination source or alternatively how much time has elapsed since the faecal matter has entered the system. Shotgun metagenomic sequencing on a subset of the samples using a portable real-time sequencer, MinION, correlated well with the amplicon sequencing data. This study demonstrates the use of DNA sequencing to detect human faecal contamination in stormwater systems and the potential of tracing faecal contamination directly in the field.

Data produced with short-read sequencing technologies result in ambiguous haplotyping and a limited capacity to investigate the full repertoire of biologically relevant forms of genetic variation. The notion of haplotype-resolved sequencing data has recently gained traction to reduce this unwanted ambiguity and enable exploration of other forms of genetic variation; beyond studies of just nucleotide polymorphisms, such as compound heterozygosity and structural variations. Here we describe Droplet Barcode Sequencing, a novel approach for creating linked-read sequencing libraries by uniquely barcoding the information within single DNA molecules in emulsion droplets, without the aid of specialty reagents or microfluidic devices. Barcode generation and template amplification is performed simultaneously in a single enzymatic reaction, greatly simplifying the workflow and minimizing assay costs compared to alternative approaches. The method has been applied to phase multiple loci targeting all exons of the highly variable Human Leukocyte Antigen A (HLA-A) gene, with DNA from eight individuals present in the same assay. Barcode-based clustering of sequencing reads confirmed analysis of over 2000 independently assayed template molecules, with an average of 753 reads in support of called polymorphisms. Our results show unequivocal characterization of all alleles present, validated by correspondence against confirmed HLA database entries and haplotyping results from previous studies., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Arsenic, a carcinogen with immunotoxic effects, is a common contaminant of drinking water and certain food worldwide. We hypothesized that chronic arsenic exposure alters gene expression, potentially by altering DNA methylation of genes encoding central components of the immune system. We therefore analyzed the transcriptomes (by RNA sequencing) and methylomes (by target-enrichment next-generation sequencing) of primary CD4-positive T cells from matched groups of four women each in the Argentinean Andes, with fivefold differences in urinary arsenic concentrations (median concentrations of urinary arsenic in the lower- and high-arsenic groups: 65 and 276 μg/l, respectively). Arsenic exposure was associated with genome-wide alterations of gene expression; principal component analysis indicated that the exposure explained 53% of the variance in gene expression among the top variable genes and 19% of 28,351 genes were differentially expressed (false discovery rate <0.05) between the exposure groups. Key genes regulating the immune system, such as tumor necrosis factor alpha and interferon gamma, as well as genes related to the NF-kappa-beta complex, were significantly downregulated in the high-arsenic group. Arsenic exposure was associated with genome-wide DNA methylation; the high-arsenic group had 3% points higher genome-wide full methylation (>80% methylation) than the lower-arsenic group. Differentially methylated regions that were hyper-methylated in the high-arsenic group showed enrichment for immune-related gene ontologies that constitute the basic functions of CD4-positive T cells, such as isotype switching and lymphocyte activation and differentiation. In conclusion, chronic arsenic exposure from drinking water was related to changes in the transcriptome and methylome of CD4-positive T cells, both genome wide and in specific genes, supporting the hypothesis that arsenic causes immunotoxicity by interfering with gene expression and regulation.

Most balanced translocations are thought to result mechanistically from nonhomologous end joining or, in rare cases of recurrent events, by nonallelic homologous recombination. Here, we use low-coverage mate pair whole-genome sequencing to fine map rearrangement breakpoint junctions in both phenotypically normal and affected translocation carriers. In total, 46 junctions from 22 carriers of balanced translocations were characterized. Genes were disrupted in 48% of the breakpoints; recessive genes in four normal carriers and known dominant intellectual disability genes in three affected carriers. Finally, seven candidate disease genes were disrupted in five carriers with neurocognitive disabilities (SVOPL, SUSD1, TOX, NCALD, SLC4A10) and one XX-male carrier with Tourette syndrome (LYPD6, GPC5). Breakpoint junction analyses revealed microhomology and small templated insertions in a substantive fraction of the analyzed translocations (17.4%; n = 4); an observation that was substantiated by reanalysis of 37 previously published translocation junctions. Microhomology associated with templated insertions is a characteristic seen in the breakpoint junctions of rearrangements mediated by error-prone replication-based repair mechanisms. Our data implicate that a mechanism involving template switching might contribute to the formation of at least 15% of the interchromosomal translocation events., (© 2016 WILEY PERIODICALS, INC.)

Pipeline tools are becoming increasingly important within the field of bioinformatics. Using a pipeline manager to manage and run workflows comprised of multiple tools reduces workload and makes analysis results more reproducible. Existing tools require significant work to install and get running, typically needing pipeline scripts to be written from scratch before running any analysis. We present Cluster Flow, a simple and flexible bioinformatics pipeline tool designed to be quick and easy to install. Cluster Flow comes with 40 modules for common NGS processing steps, ready to work out of the box. Pipelines are assembled using these modules with a simple syntax that can be easily modified as required. Core helper functions automate many common NGS procedures, making running pipelines simple. Cluster Flow is available with an GNU GPLv3 license on GitHub. Documentation, examples and an online demo are available at http://clusterflow.io., Competing Interests: Competing interests: No competing interests were disclosed.

Motivation: Fast and accurate quality control is essential for studies involving next-generation sequencing data. Whilst numerous tools exist to quantify QC metrics, there is no common approach to flexibly integrate these across tools and large sample sets. Assessing analysis results across an entire project can be time consuming and error prone; batch effects and outlier samples can easily be missed in the early stages of analysis., Results: We present MultiQC, a tool to create a single report visualising output from multiple tools across many samples, enabling global trends and biases to be quickly identified. MultiQC can plot data from many common bioinformatics tools and is built to allow easy extension and customization., Availability and Implementation: MultiQC is available with an GNU GPLv3 license on GitHub, the Python Package Index and Bioconda. Documentation and example reports are available at http://multiqc.info, Contact: phil.ewels@scilifelab.se., (© The Author 2016. Published by Oxford University Press.)

To improve the epigenomic analysis of tissues rich in 5-hydroxymethylcytosine (hmC), we developed a novel protocol called TAB-Methyl-SEQ, which allows for single base resolution profiling of both hmC and 5-methylcytosine by targeted next-generation sequencing. TAB-Methyl-SEQ data were extensively validated by a set of five methodologically different protocols. Importantly, these extensive cross-comparisons revealed that protocols based on Tet1-assisted bisulfite conversion provided more precise hmC values than TrueMethyl-based methods. A total of 109 454 CpG sites were analyzed by TAB-Methyl-SEQ for mC and hmC in 188 genes from 20 different adult human livers. We describe three types of variability of hepatic hmC profiles: (i) sample-specific variability at 40.8% of CpG sites analyzed, where the local hmC values correlate to the global hmC content of livers (measured by LC-MS), (ii) gene-specific variability, where hmC levels in the coding regions positively correlate to expression of the respective gene and (iii) site-specific variability, where prominent hmC peaks span only 1 to 3 neighboring CpG sites. Our data suggest that both the gene- and site-specific components of hmC variability might contribute to the epigenetic control of hepatic genes. The protocol described here should be useful for targeted DNA analysis in a variety of applications., (© The Author 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Bisulfite treatment of DNA followed by sequencing (BS-seq) has become a standard technique in epigenetic studies, providing researchers with tools for generating single-base resolution maps of whole methylomes. Aligning bisulfite-treated reads, however, is a computationally difficult task: bisulfite treatment decreases the (lexical) complexity of low-methylated genomic regions, and C-to-T mismatches may reflect cytosine unmethylation rather than SNPs or sequencing errors. Further challenges arise both during and after the alignment phase: data structures used by the aligner should be fast and should fit into main memory, and the methylation-caller output should be somehow compressed, due to its significant size., Methods: As far as data structures employed to align bisulfite-treated reads are concerned, solutions proposed in the literature can be roughly grouped into two main categories: those storing pointers at each text position (e.g. hash tables, suffix trees/arrays), and those using the information-theoretic minimum number of bits (e.g. FM indexes and compressed suffix arrays). The former are fast and memory consuming. The latter are much slower and light. In this paper, we try to close this gap proposing a data structure for aligning bisulfite-treated reads which is at the same time fast, light, and very accurate. We reach this objective by combining a recent theoretical result on succinct hashing with a bisulfite-aware hash function. Furthermore, the new versions of the tools implementing our ideas|the aligner ERNE-BS5 2 and the caller ERNE-METH 2|have been extended with increased downstream compatibility (EPP/Bismark cov output formats), output compression, and support for target enrichment protocols., Results: Experimental results on public and simulated WGBS libraries show that our algorithmic solution is a competitive tradeoff between hash-based and BWT-based indexes, being as fast and accurate as the former, and as memory-efficient as the latter., Conclusions: The new functionalities of our bisulfite aligner and caller make it a fast and memory efficient tool, useful to analyze big datasets with little computational resources, to easily process target enrichment data, and produce statistics such as protocol efficiency and coverage as a function of the distance from target regions.

In pulmonary sarcoidosis, CD4(+) T-cells expressing T-cell receptor Vα2.3 accumulate in the lungs of HLA-DRB1*03(+) patients. To investigate T-cell receptor-HLA-DRB1*03 interactions underlying recognition of hitherto unknown antigens, we performed detailed analyses of T-cell receptor expression on bronchoalveolar lavage fluid CD4(+) T-cells from sarcoidosis patients.Pulmonary sarcoidosis patients (n=43) underwent bronchoscopy with bronchoalveolar lavage. T-cell receptor α and β chains of CD4(+) T-cells were analysed by flow cytometry, DNA-sequenced, and three-dimensional molecular models of T-cell receptor-HLA-DRB1*03 complexes generated.Simultaneous expression of Vα2.3 with the Vβ22 chain was identified in the lungs of all HLA-DRB1*03(+) patients. Accumulated Vα2.3/Vβ22-expressing T-cells were highly clonal, with identical or near-identical Vα2.3 chain sequences and inter-patient similarities in Vβ22 chain amino acid distribution. Molecular modelling revealed specific T-cell receptor-HLA-DRB1*03-peptide interactions, with a previously identified, sarcoidosis-associated vimentin peptide, (Vim)429-443 DSLPLVDTHSKRTLL, matching both the HLA peptide-binding cleft and distinct T-cell receptor features perfectly.We demonstrate, for the first time, the accumulation of large clonal populations of specific Vα2.3/Vβ22 T-cell receptor-expressing CD4(+) T-cells in the lungs of HLA-DRB1*03(+) sarcoidosis patients. Several distinct contact points between Vα2.3/Vβ22 receptors and HLA-DRB1*03 molecules suggest presentation of prototypic vimentin-derived peptides., (Copyright ©ERS 2016.)

Background: It 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., Methods: In 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., Results: We 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.

Restriction enzymes that recognize specific sequences but cleave unknown sequence outside the recognition site are extensively utilized tools in molecular biology. Despite this, systematic functional categorization of cleavage performance has largely been lacking. We established a simple and automatable model system to assay cleavage distance variation (termed slippage) and the sequence dependence thereof. We coupled this to massively parallel sequencing in order to provide sensitive and accurate measurement. With this system 14 enzymes were assayed (AcuI, BbvI, BpmI, BpuEI, BseRI, BsgI, Eco57I, Eco57MI, EcoP15I, FauI, FokI, GsuI, MmeI and SmuI). We report significant variation of slippage ranging from 1-54%, variations in sequence context dependence, as well as variation between isoschizomers. We believe this largely overlooked property of enzymes with shifted cleavage would benefit from further large scale classification and engineering efforts seeking to improve performance. The gained insights of in-vitro performance may also aid the in-vivo understanding of these enzymes.

Dictyostelium intermediate repeat sequence 1 (DIRS-1) is the founding member of a poorly characterized class of retrotransposable elements that contain inverse long terminal repeats and tyrosine recombinase instead of DDE-type integrase enzymes. In Dictyostelium discoideum, DIRS-1 forms clusters that adopt the function of centromeres, rendering tight retrotransposition control critical to maintaining chromosome integrity. We report that in deletion strains of the RNA-dependent RNA polymerase RrpC, full-length and shorter DIRS-1 messenger RNAs are strongly enriched. Shorter versions of a hitherto unknown long non-coding RNA in DIRS-1 antisense orientation are also enriched in rrpC- strains. Concurrent with the accumulation of long transcripts, the vast majority of small (21 mer) DIRS-1 RNAs vanish in rrpC- strains. RNASeq reveals an asymmetric distribution of the DIRS-1 small RNAs, both along DIRS-1 and with respect to sense and antisense orientation. We show that RrpC is required for post-transcriptional DIRS-1 silencing and also for spreading of RNA silencing signals. Finally, DIRS-1 mis-regulation in the absence of RrpC leads to retrotransposon mobilization. In summary, our data reveal RrpC as a key player in the silencing of centromeric retrotransposon DIRS-1. RrpC acts at the post-transcriptional level and is involved in spreading of RNA silencing signals, both in the 5' and 3' directions.

Exome sequence capture and massively parallel sequencing can be combined to achieve inexpensive and rapid global analyses of the functional sections of the genome. The difficulties of working with relatively small quantities of genetic material, as may be necessary when sharing tumor biopsies between collaborators for instance, can be overcome using whole genome amplification. However, the potential drawbacks of using a whole genome amplification technology based on random primers in combination with sequence capture followed by massively parallel sequencing have not yet been examined in detail, especially in the context of mutation discovery in tumor material. In this work, we compare mutations detected in sequence data for unamplified DNA, whole genome amplified DNA, and RNA originating from the same tumor tissue samples from 16 patients diagnosed with non-small cell lung cancer. The results obtained provide a comprehensive overview of the merits of these techniques for mutation analysis. We evaluated the identified genetic variants, and found that most (74%) of them were observed in both the amplified and the unamplified sequence data. Eighty-nine percent of the variations found by WGA were shared with unamplified DNA. We demonstrate a strategy for avoiding allelic bias by including RNA-sequencing information.

Conifers have dominated forests for more than 200 million years and are of huge ecological and economic importance. Here we present the draft assembly of the 20-gigabase genome of Norway spruce (Picea abies), the first available for any gymnosperm. The number of well-supported genes (28,354) is similar to the >100 times smaller genome of Arabidopsis thaliana, and there is no evidence of a recent whole-genome duplication in the gymnosperm lineage. Instead, the large genome size seems to result from the slow and steady accumulation of a diverse set of long-terminal repeat transposable elements, possibly owing to the lack of an efficient elimination mechanism. Comparative sequencing of Pinus sylvestris, Abies sibirica, Juniperus communis, Taxus baccata and Gnetum gnemon reveals that the transposable element diversity is shared among extant conifers. Expression of 24-nucleotide small RNAs, previously implicated in transposable element silencing, is tissue-specific and much lower than in other plants. We further identify numerous long (>10,000 base pairs) introns, gene-like fragments, uncharacterized long non-coding RNAs and short RNAs. This opens up new genomic avenues for conifer forestry and breeding.

Here we demonstrate the use of short-read massive sequencing systems to in effect achieve longer read lengths through hierarchical molecular tagging. We show how indexed and PCR-amplified targeted libraries are degraded, sub-sampled and arrested at timed intervals to achieve pools of differing average length, each of which is indexed with a new tag. By this process, indices of sample origin, molecular origin, and degree of degradation is incorporated in order to achieve a nested hierarchical structure, later to be utilized in the data processing to order the reads over a longer distance than the sequencing system originally allows. With this protocol we show how continuous regions beyond 3000 bp can be decoded by an Illumina sequencing system, and we illustrate the potential applications by calling variants of the lambda genome, analysing TP53 in cancer cell lines, and targeting a variable canine mitochondrial region.

High-throughput sequencing methods have become invaluable for detection and analysis of small RNAs. The results are millions of sequences that need to be carefully analyzed by computational methods and preferentially verified by different experimental techniques. Here we describe how to use high-throughput sequencing followed by bioinformatics and northern blot to identify one particular class of small RNA, microRNAs.

Motivation: New generation sequencing technologies producing increasingly complex datasets demand new efficient and specialized sequence analysis algorithms. Often, it is only the 'novel' sequences in a complex dataset that are of interest and the superfluous sequences need to be removed., Results: A novel algorithm, fast and accurate classification of sequences (FACSs), is introduced that can accurately and rapidly classify sequences as belonging or not belonging to a reference sequence. FACS was first optimized and validated using a synthetic metagenome dataset. An experimental metagenome dataset was then used to show that FACS achieves comparable accuracy as BLAT and SSAHA2 but is at least 21 times faster in classifying sequences., Availability: Source code for FACS, Bloom filters and MetaSim dataset used is available at http://facs.biotech.kth.se. The Bloom::Faster 1.6 Perl module can be downloaded from CPAN at http://search.cpan.org/ approximately palvaro/Bloom-Faster-1.6/, Contacts: henrik.stranneheim@biotech.kth.se; joakiml@biotech.kth.se, Supplementary Information: Supplementary data are available at Bioinformatics online.

We report on the incorporation of the Visual DNA concept in a genotyping assay as a simple and straightforward detection tool. The principle of trapping streptavidin-coated superparamagnetic beads of micrometer size for visualization of genetic variances is used for PrASE-based detection of a panel of mutations in the severe and common genetic disorder of cystic fibrosis. The method allows a final investigation of genotypes by the naked eye and the output is easily documented using a regular hand-held device with an integrated digital camera. A number of samples were run through the assay, showing rapid and accurate detection using superparamagnetic beads and an off-the-shelf neodymium magnet. The assay emphasizes the power of Visual DNA and demonstrates the potential value of the method in future point-of-care tests.

The genetic background of cutaneous malignant melanoma (CMM) includes both germ line aberrations in high-penetrance genes, like CDKN2A, and allelic variation in low-penetrance genes like the melanocortin-1 receptor gene, MC1R. Red-hair colour associated MC1R alleles (RHC) have been associated with red hair, fair skin and risk of CMM. We investigated MC1R and CDKN2A variation in relation to phenotype, clinical factors and CMM risk in the Swedish population. The study cohort consisted of sporadic primary melanoma patients, familial melanoma patients and a control group. An allele-dose dependent increase in melanoma risk for carriers of variant MC1R alleles (after adjusting for phenotype), with an elevated risk among familial CMM patients, was observed. This elevated risk was found to be significantly associated with an increased frequency of dysplastic nevi (DN) among familial patients compared to sporadic patients. MC1R variation was found to be less frequent among acral lentiginous melanomas (ALM) and dependent on tumour localisation. No association was found between CDKN2A gene variants and general melanoma risk. Two new variants in the POMC gene were identified in red haired individuals without RHC alleles.

Over the last few years, several initiatives have described efforts to combine previously invented techniques in molecular biology with parallel detection principles to sequence or genotype DNA signatures. The Infinium system from Illumina and the Affymetrix GeneChips are two systems suitable for whole-genome scoring of variable positions. However, directed candidate-gene approaches are more cost effective and several academic groups and the private sector provide techniques with moderate typing throughput combined with large sample capacity suiting these needs. Recently, whole-genome sequencing platforms based on the sequencing-by-synthesis principle were presented by 454 Life Sciences and Solexa, showing great potential as alternatives to conventional genotyping approaches. In addition to these sequencing initiatives, many efforts are pursuing novel ideas to facilitate fast and cost-effective whole genome sequencing, such as ligation-based sequencing. Reliable methods for routine re-sequencing of human genomes as a tool for personalized medicine, however, remain to be developed.

Background: There is an imperative need for SNP genotyping technologies that are cost-effective per sample with retained high accuracy, throughput and flexibility. We have developed a microarray-based technique and compared it to Pyrosequencing. In the protease-mediated allele-specific extension (PrASE), the protease constrains the elongation reaction and thus prevents incorrect nucleotide incorporation to mismatched 3'-termini primers., Results: The assay is automated for 48 genotyping reactions in parallel followed by a tag-microarray detection system. A script automatically visualizes the results in cluster diagrams and assigns the genotypes. Ten polymorphic positions suggested as prothrombotic genetic variations were analyzed with Pyrosequencing and PrASE technologies in 442 samples and 99.8 % concordance was achieved. In addition to accuracy, the robustness and reproducibility of the technique has been investigated., Conclusion: The results of this study strongly indicate that the PrASE technology can offer significant improvements in terms of accuracy and robustness and thereof increased number of typeable SNPs.

A method is described for HPV genotyping based on multiplex competitive hybridization (MUCH) combined with apyrase mediated allele-specific extension (AMASE). Two type-specific oligonucleotides were designed for each of the 23 investigated HPV types and directed towards two highly inter-type heterogeneous regions. The type-specific oligonucleotides were allowed to compete in the hybridization to an immobilized template resulting in a highly specific hybridization process. To increase further the specificity, a second step of type discrimination was used in which specific extension of 3'-termini matched oligonucleotides was performed. The 46 type-specific oligonucleotides each had a unique tag sequence to allow detection via an array of oligonucleotides complementary to the tags. To evaluate the genotyping assay, a total of 92 HPV positive samples were tested in this study. Twelve had double infections and five had three to five coexisting HPV types. The results show that MUCH-AMASE can readily detect multiple infections, whereas conventional dideoxy sequencing resulted in ambiguous sequence. Four samples with three to five genotypes detected were cloned and individual clones were sequenced. The cloning procedure verified the MUCH-AMASE results with indications that we can find minor infections (<2% relative amounts). We can thus conclude that the developed assay is highly sensitive, with improved throughput and with excellent possibility to detect multiple infections.

Here, we present a novel method for SNP genotyping based on protease-mediated allele-specific primer extension (PrASE), where the two allele-specific extension primers only differ in their 3'-positions. As reported previously [Ahmadian,A., Gharizadeh,B., O'Meara,D., Odeberg,J. and Lundeberg,J. (2001), Nucleic Acids Res., 29, e121], the kinetics of perfectly matched primer extension is faster than mismatched primer extension. In this study, we have utilized this difference in kinetics by adding protease, a protein-degrading enzyme, to discriminate between the extension reactions. The competition between the polymerase activity and the enzymatic degradation yields extension of the perfectly matched primer, while the slower extension of mismatched primer is eliminated. To allow multiplex and simultaneous detection of the investigated single nucleotide polymorphisms (SNPs), each extension primer was given a unique signature tag sequence on its 5' end, complementary to a tag on a generic array. A multiplex nested PCR with 13 SNPs was performed in a total of 36 individuals and their alleles were scored. To demonstrate the improvements in scoring SNPs by PrASE, we also genotyped the individuals without inclusion of protease in the extension. We conclude that the developed assay is highly allele-specific, with excellent multiplex SNP capabilities.

Alterations in the p53 tumor suppressor gene are important events in many cases of human cancers. We have developed a novel microarray based approach for re-sequencing and mutation detection of the p53 gene. The method facilitates rapid and simple scanning of the target gene sequence and could be expanded to include other candidate cancer genes. The methodology employs the previously described apyrase-mediated allele-specific extension reaction (AMASE). In order to re-sequence the selected region, four extension oligonucleotides with different 3'-termini were used for each base position and they were covalently attached to the glass slide's surface. The amplified single-stranded DNA templates were then hybridized to the array followed by in situ extension with fluorescently labeled dNTPs in the presence of apyrase. The model system used was based on analysis of a 15 bp stretch in exon 5 of the p53 gene. Mutations were scored as allelic fractions calculated as (wt)/(wt + mut) signals. When apyrase was included in the extension reactions of wild type templates, the mean allelic fraction was 0.96. When apyrase was excluded with the same wild type templates, significantly lower allelic fractions were obtained. Two 60-mer synthetic oligonucleotides were used to establish the detectable amount of mutations with AMASE and a clear distinction between all the points could be made. Several samples from different stages of skin malignancies were also analyzed. The results from this study imply the possibility to efficiently and accurately re-sequence the entire p53 gene with AMASE technology.

Detection and identification of microbial pathogens are important for disease diagnosis, treatment and prophylaxis measurements. By introducing an innovative technique, we show a robust, reliable and accurate microarray-based method for identification of microbial pathogens. The technique utilizes a unique combination of multiplex competitive hybridization, which enhances hybridization accuracy of oligonucleotides to the specific target, and apyrase-mediated allele-specific extension, which improves specific extension. As a model system, different clinically relevant human papillomaviruses were selected for this study. The method generated accurate results and proves to be promising for specific and correct microbial and viral typing.