Biostatistical Aspects of Whole Genome Sequencing Studies: Preprocessing and Quality Control.

Rapid advances in high-throughput DNA sequencing technologies have enabled large-scale whole genome sequencing (WGS) studies. Before performing association analysis between phenotypes and genotypes, preprocessing and quality control (QC) of the raw sequence data need to be performed. Because many biostatisticians have not been working with WGS data so far, we first sketch Illumina's short-read sequencing technology. Second, we explain the general preprocessing pipeline for WGS studies. Third, we provide an overview of important QC metrics, which are applied to WGS data: on the raw data, after mapping and alignment, after variant calling, and after multisample variant calling. Fourth, we illustrate the QC with the data from the GENEtic SequencIng Study Hamburg-Davos (GENESIS-HD), a study involving more than 9000 human whole genomes. All samples were sequenced on an Illumina NovaSeq 6000 with an average coverage of 35× using a PCR-free protocol. For QC, one genome in a bottle (GIAB) trio was sequenced in four replicates, and one GIAB sample was successfully sequenced 70 times in different runs. Fifth, we provide empirical data on the compression of raw data using the DRAGEN original read archive (ORA). The most important quality metrics in the application were genetic similarity, sample cross-contamination, deviations from the expected Het/Hom ratio, relatedness, and coverage. The compression ratio of the raw files using DRAGEN ORA was 5.6:1, and compression time was linear by genome coverage. In summary, the preprocessing, joint calling, and QC of large WGS studies are feasible within a reasonable time, and efficient QC procedures are readily available.

Patchwork: Alignment-Based Retrieval and Concatenation of Phylogenetic Markers from Genomic Data.

Low-coverage whole-genome sequencing (also known as "genome skimming") is becoming an increasingly affordable approach to large-scale phylogenetic analyses. While already routinely used to recover organellar genomes, genome skimming is rather rarely utilized for recovering single-copy nuclear markers. One reason might be that only few tools exist to work with this data type within a phylogenomic context, especially to deal with fragmented genome assemblies. We here present a new software tool called Patchwork for mining phylogenetic markers from highly fragmented short-read assemblies as well as directly from sequence reads. Patchwork is an alignment-based tool that utilizes the sequence aligner DIAMOND and is written in the programming language Julia. Homologous regions are obtained via a sequence similarity search, followed by a "hit stitching" phase, in which adjacent or overlapping regions are merged into a single unit. The novel sliding window algorithm trims away any noncoding regions from the resulting sequence. We demonstrate the utility of Patchwork by recovering near-universal single-copy orthologs within a benchmarking study, and we additionally assess the performance of Patchwork in comparison with other programs. We find that Patchwork allows for accurate retrieval of (putatively) single-copy genes from genome skimming data sets at different sequencing depths with high computational speed, outperforming existing software targeting similar tasks. Patchwork is released under the GNU General Public License version 3. Installation instructions, additional documentation, and the source code itself are all available via GitHub at https://github.com/fethalen/Patchwork.

Evolutionary dynamics of whole-body regeneration across planarian flatworms.

Regenerative abilities vary dramatically across animals. Even amongst planarian flatworms, well-known for complete regeneration from tiny body fragments, some species have restricted regeneration abilities while others are almost entirely regeneration incompetent. Here, we assemble a diverse live collection of 40 planarian species to probe the evolution of head regeneration in the group. Combining quantification of species-specific head-regeneration abilities with a comprehensive transcriptome-based phylogeny reconstruction, we show multiple independent transitions between robust whole-body regeneration and restricted regeneration in freshwater species. RNA-mediated genetic interference inhibition of canonical Wnt signalling in RNA-mediated genetic interference-sensitive species bypassed all head-regeneration defects, suggesting that the Wnt pathway is linked to the emergence of planarian regeneration defects. Our finding that Wnt signalling has multiple roles in the reproductive system of the model species Schmidtea mediterranea raises the possibility that a trade-off between egg-laying, asexual reproduction by fission/regeneration and Wnt signalling drives regenerative trait evolution. Although quantitative comparisons of Wnt signalling levels, yolk content and reproductive strategy across our species collection remained inconclusive, they revealed divergent Wnt signalling roles in the reproductive system of planarians. Altogether, our study establishes planarians as a model taxon for comparative regeneration research and presents a framework for the mechanistic evolution of regenerative abilities.