Sustainable data analysis with Snakemake.

Data analysis often entails a multitude of heterogeneous steps, from the application of various command line tools to the usage of scripting languages like R or Python for the generation of plots and tables. It is widely recognized that data analyses should ideally be conducted in a reproducible way. Reproducibility enables technical validation and regeneration of results on the original or even new data. However, reproducibility alone is by no means sufficient to deliver an analysis that is of lasting impact (i.e., sustainable) for the field, or even just one research group. We postulate that it is equally important to ensure adaptability and transparency. The former describes the ability to modify the analysis to answer extended or slightly different research questions. The latter describes the ability to understand the analysis in order to judge whether it is not only technically, but methodologically valid. Here, we analyze the properties needed for a data analysis to become reproducible, adaptable, and transparent. We show how the popular workflow management system Snakemake can be used to guarantee this, and how it enables an ergonomic, combined, unified representation of all steps involved in data analysis, ranging from raw data processing, to quality control and fine-grained, interactive exploration and plotting of final results.

The combination of WGS and RNA-Seq is superior to conventional diagnostic tests in multiple myeloma: Ready for prime time?

The diagnosis and risk stratification of multiple myeloma (MM) is based on clinical and cytogenetic tests. Magnetic CD138 enrichment followed by interphase FISH (fluorescence in situ hybridisation) is the gold standard to identify prognostic translocations and copy number alterations (CNA). Although clinical implications of gene expression profiling (GEP) or panel based sequencing results are evident, those tests have not yet reached routine clinical application. We set up a single workflow to analyse MM of 211 patients at first diagnosis by whole genome sequencing (WGS) and RNA-Seq and validate the results by FISH analysis. We observed a 96% concordance of FISH and WGS results when assessing translocations involving the IGH locus and an overall concordance of FISH and WGS of 92% when assessing CNA. WGS analysis resulted in the identification of 17 additional MYC-translocations that were missed by FISH analysis. RNA-Seq followed by supervised clustering grouped patients in their expected genetically defined subgroup and prompted the assessment of WGS data in cases that were not congruent with FISH. This allowed the identification of additional IGH-translocations and hyperdiploid cases. We show the reliability of WGS an RNA-Seq in a clinical setting, which is a prerequisite for a novel routine diagnostic test.

Durum wheat genome highlights past domestication signatures and future improvement targets.

The domestication of wild emmer wheat led to the selection of modern durum wheat, grown mainly for pasta production. We describe the 10.45 gigabase (Gb) assembly of the genome of durum wheat cultivar Svevo. The assembly enabled genome-wide genetic diversity analyses revealing the changes imposed by thousands of years of empirical selection and breeding. Regions exhibiting strong signatures of genetic divergence associated with domestication and breeding were widespread in the genome with several major diversity losses in the pericentromeric regions. A locus on chromosome 5B carries a gene encoding a metal transporter (TdHMA3-B1) with a non-functional variant causing high accumulation of cadmium in grain. The high-cadmium allele, widespread among durum cultivars but undetected in wild emmer accessions, increased in frequency from domesticated emmer to modern durum wheat. The rapid cloning of TdHMA3-B1 rescues a wild beneficial allele and demonstrates the practical use of the Svevo genome for wheat improvement.

Chromosome-scale comparative sequence analysis unravels molecular mechanisms of genome dynamics between two wheat cultivars.

BACKGROUND: Recent improvements in DNA sequencing and genome scaffolding have paved the way to generate high-quality de novo assemblies of pseudomolecules representing complete chromosomes of wheat and its wild relatives. These assemblies form the basis to compare the dynamics of wheat genomes on a megabase scale. RESULTS: Here, we provide a comparative sequence analysis of the 700-megabase chromosome 2D between two bread wheat genotypes-the old landrace Chinese Spring and the elite Swiss spring wheat line 'CH Campala Lr22a'. Both chromosomes were assembled into megabase-sized scaffolds. There is a high degree of sequence conservation between the two chromosomes. Analysis of large structural variations reveals four large indels of more than 100 kb. Based on the molecular signatures at the breakpoints, unequal crossing over and double-strand break repair were identified as the molecular mechanisms that caused these indels. Three of the large indels affect copy number of NLRs, a gene family involved in plant immunity. Analysis of SNP density reveals four haploblocks of 4, 8, 9 and 48 Mb with a 35-fold increased SNP density compared to the rest of the chromosome. Gene content across the two chromosomes was highly conserved. Ninety-nine percent of the genic sequences were present in both genotypes and the fraction of unique genes ranged from 0.4 to 0.7%. CONCLUSIONS: This comparative analysis of two high-quality chromosome assemblies enabled a comprehensive assessment of large structural variations and gene content. The insight obtained from this analysis will form the basis of future wheat pan-genome studies.

Combining RNA-seq data and homology-based gene prediction for plants, animals and fungi.

BACKGROUND: Genome annotation is of key importance in many research questions. The identification of protein-coding genes is often based on transcriptome sequencing data, ab-initio or homology-based prediction. Recently, it was demonstrated that intron position conservation improves homology-based gene prediction, and that experimental data improves ab-initio gene prediction. RESULTS: Here, we present an extension of the gene prediction program GeMoMa that utilizes amino acid sequence conservation, intron position conservation and optionally RNA-seq data for homology-based gene prediction. We show on published benchmark data for plants, animals and fungi that GeMoMa performs better than the gene prediction programs BRAKER1, MAKER2, and CodingQuarry, and purely RNA-seq-based pipelines for transcript identification. In addition, we demonstrate that using multiple reference organisms may help to further improve the performance of GeMoMa. Finally, we apply GeMoMa to four nematode species and to the recently published barley reference genome indicating that current annotations of protein-coding genes may be refined using GeMoMa predictions. CONCLUSIONS: GeMoMa might be of great utility for annotating newly sequenced genomes but also for finding homologs of a specific gene or gene family. GeMoMa has been published under GNU GPL3 and is freely available at http://www.jstacs.de/index.php/GeMoMa .

Genome sequence of the progenitor of the wheat D genome Aegilops tauschii.

Aegilops tauschii is the diploid progenitor of the D genome of hexaploid wheat (Triticum aestivum, genomes AABBDD) and an important genetic resource for wheat. The large size and highly repetitive nature of the Ae. tauschii genome has until now precluded the development of a reference-quality genome sequence. Here we use an array of advanced technologies, including ordered-clone genome sequencing, whole-genome shotgun sequencing, and BioNano optical genome mapping, to generate a reference-quality genome sequence for Ae. tauschii ssp. strangulata accession AL8/78, which is closely related to the wheat D genome. We show that compared to other sequenced plant genomes, including a much larger conifer genome, the Ae. tauschii genome contains unprecedented amounts of very similar repeated sequences. Our genome comparisons reveal that the Ae. tauschii genome has a greater number of dispersed duplicated genes than other sequenced genomes and its chromosomes have been structurally evolving an order of magnitude faster than those of other grass genomes. The decay of colinearity with other grass genomes correlates with recombination rates along chromosomes. We propose that the vast amounts of very similar repeated sequences cause frequent errors in recombination and lead to gene duplications and structural chromosome changes that drive fast genome evolution.

Light and Plastid Signals Regulate Different Sets of Genes in the Albino Mutant Pap7-1.

Plants possessing dysfunctional plastids due to defects in pigment biosynthesis or translation are known to repress photosynthesis-associated nuclear genes via retrograde signals from the disturbed organelles toward the nucleus. These signals are thought to be essential for proper biogenesis and function of the plastid. Mutants lacking plastid-encoded RNA polymerase-associated proteins (PAPs) display a genetic arrest in eoplast-chloroplast transition leading to an albino phenotype in the light. Retrograde signaling in these mutants, therefore, could be expected to be similar as under conditions inducing plastid dysfunction. To answer this question, we performed plastome- and genomewide array analyses in the pap7-1 mutant of Arabidopsis (Arabidopsis thaliana). In parallel, we determined the potential overlap with light-regulated expression networks. To this end, we performed a comparative expression profiling approach using light- and dark-grown wild-type plants as relative control for the expression profiles obtained from light-grown pap7-1 mutants. Our data indicate a specific impact of retrograde signals on metabolism-related genes in pap7-1 mutants reflecting the starvation situation of the albino seedlings. In contrast, light regulation of PhANGs and other nuclear gene groups appears to be fully functional in this mutant, indicating that a block in chloroplast biogenesis per se does not repress expression of them as suggested by earlier studies. Only genes for light harvesting complex proteins displayed a significant repression indicating an exclusive retrograde impact on this gene family. Our results indicate that chloroplasts and arrested plastids each emit specific signals that control different target gene modules both in positive and negative manner.

Wild emmer genome architecture and diversity elucidate wheat evolution and domestication.

Wheat (Triticum spp.) is one of the founder crops that likely drove the Neolithic transition to sedentary agrarian societies in the Fertile Crescent more than 10,000 years ago. Identifying genetic modifications underlying wheat's domestication requires knowledge about the genome of its allo-tetraploid progenitor, wild emmer (T. turgidum ssp. dicoccoides). We report a 10.1-gigabase assembly of the 14 chromosomes of wild tetraploid wheat, as well as analyses of gene content, genome architecture, and genetic diversity. With this fully assembled polyploid wheat genome, we identified the causal mutations in Brittle Rachis 1 (TtBtr1) genes controlling shattering, a key domestication trait. A study of genomic diversity among wild and domesticated accessions revealed genomic regions bearing the signature of selection under domestication. This reference assembly will serve as a resource for accelerating the genome-assisted improvement of modern wheat varieties.

A chromosome conformation capture ordered sequence of the barley genome.

Cereal grasses of the Triticeae tribe have been the major food source in temperate regions since the dawn of agriculture. Their large genomes are characterized by a high content of repetitive elements and large pericentromeric regions that are virtually devoid of meiotic recombination. Here we present a high-quality reference genome assembly for barley (Hordeum vulgare L.). We use chromosome conformation capture mapping to derive the linear order of sequences across the pericentromeric space and to investigate the spatial organization of chromatin in the nucleus at megabase resolution. The composition of genes and repetitive elements differs between distal and proximal regions. Gene family analyses reveal lineage-specific duplications of genes involved in the transport of nutrients to developing seeds and the mobilization of carbohydrates in grains. We demonstrate the importance of the barley reference sequence for breeding by inspecting the genomic partitioning of sequence variation in modern elite germplasm, highlighting regions vulnerable to genetic erosion.

Towards a whole-genome sequence for rye (Secale cereale L.).

We report on a whole-genome draft sequence of rye (Secale cereale L.). Rye is a diploid Triticeae species closely related to wheat and barley, and an important crop for food and feed in Central and Eastern Europe. Through whole-genome shotgun sequencing of the 7.9-Gbp genome of the winter rye inbred line Lo7 we obtained a de novo assembly represented by 1.29 million scaffolds covering a total length of 2.8 Gbp. Our reference sequence represents nearly the entire low-copy portion of the rye genome. This genome assembly was used to predict 27 784 rye gene models based on homology to sequenced grass genomes. Through resequencing of 10 rye inbred lines and one accession of the wild relative S. vavilovii, we discovered more than 90 million single nucleotide variants and short insertions/deletions in the rye genome. From these variants, we developed the high-density Rye600k genotyping array with 600 843 markers, which enabled anchoring the sequence contigs along a high-density genetic map and establishing a synteny-based virtual gene order. Genotyping data were used to characterize the diversity of rye breeding pools and genetic resources, and to obtain a genome-wide map of selection signals differentiating the divergent gene pools. This rye whole-genome sequence closes a gap in Triticeae genome research, and will be highly valuable for comparative genomics, functional studies and genome-based breeding in rye.

The general composition of the faecal virome of pigs depends on age, but not on feeding with a probiotic bacterium.

BACKGROUND: The pig faecal virome, which comprises the community of viruses present in pig faeces, is complex and consists of pig viruses, bacteriophages, transiently passaged plant viruses and other minor virus species. Only little is known about factors influencing its general composition. Here, the effect of the probiotic bacterium Enterococcus faecium (E. faecium) NCIMB 10415 on the pig faecal virome composition was analysed in a pig feeding trial with sows and their piglets, which received either the probiotic bacterium or not. RESULTS: From 8 pooled faecal samples derived from the feeding trial, DNA and RNA virus particles were prepared and subjected to process-controlled Next Generation Sequencing resulting in 390,650 sequence reads. In average, 14% of the reads showed significant sequence identities to known viruses. The percentage of detected mammalian virus sequences was highest (55-77%) in the samples of the youngest piglets and lowest (8-10%) in the samples of the sows. In contrast, the percentage of bacteriophage sequences increased from 22-44% in the youngest piglets to approximately 90% in the sows. The dominating mammalian viruses differed remarkably among 12 day-old piglets (kobuvirus), 54 day-old piglets (boca-, dependo- and pig stool-associated small circular DNA virus [PigSCV]) and the sows (PigSCV, circovirus and "circovirus-like" viruses CB-A and RW-A). In addition, the Shannon index, which reflects the diversity of sequences present in a sample, was generally higher for the sows as compared to the piglets. No consistent differences in the virome composition could be identified between the viromes of the probiotic bacterium-treated group and the control group. CONCLUSION: The analysis indicates that the pig faecal virome shows a high variability and that its general composition is mainly dependent on the age of the pigs. Changes caused by feeding with the probiotic bacterium E. faecium could not be demonstrated using the applied metagenomics method.