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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/.

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.

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. The draft genome of the Norway spruce ( P. abies ) is presented; this is the first gymnosperm genome to be sequenced and reveals a large genome size (20 Gb) resulting from the accumulation of transposable elements, and comparative sequencing of five other gymnosperm genomes provides insights into conifer genome evolution. Conifer genome looms large The first draft gymnosperm genome, that of a Norway spruce ( Picea abies ), is published this week by the Spruce Genome Project consortium. The genome is from a tree originally collected in 1959 in eastern Jämtland, central Sweden. At 20 gigabases, the genome is more than a hundred times larger than that of the model plant species Arabidopsis , but the two contain a similar number of genes. The large genome size is the result of an accumulation of transposable elements. Comparative sequencing of five further gymnosperm genomes suggests that transposable element diversity is shared among extant conifers. The sequence data are available for public access from the ConGenIE website ( http://congenie.org/ ).

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.

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/.

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.

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.

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.

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.

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.