RLM: fast and simplified extraction of read-level methylation metrics from bisulfite sequencing data

Bisulfite sequencing data provide value beyond the straightforward methylation assessment by analyzing single-read patterns. Over the past years, various metrics have been established to explore this layer of information. However, limited compatibility with alignment tools, reference genomes or the measurements they provide present a bottleneck for most groups to routinely perform read-level analysis. To address this, we developed RLM, a fast and scalable tool for the computation of several frequently used read-level methylation statistics. RLM supports standard alignment tools, works independently of the reference genome and handles most sequencing experiment designs. RLM can process large input files with a billion reads in just a few hours on common workstations

Genome Analysis Methods using Long Read Nanopore Sequencing

Third-generation long-read technologies denote the latest progression in high throughput DNA and RNA sequence analysis. Complementing the widespread second-generation short-read platforms, long-read sequencing adds unique application opportunities by generating previously unattainable read lengths. Despite the remaining higher error rate compared to short reads, single-molecule real-time sequencing (SMRT) and nanopore sequencing advanced to be state-of-the-art for \textit{de-novo} genome assemblies and identification of structural variants. Continuous throughput and accuracy improvements lead to development of novel methods and applications at a fast pace. We identify major application fields and key bioinformatic software for long-read sequencing data analysis by employing a data driven literature research. The integration of citations and keywords into a literature graph provides a scaling approach to analyze an exponentially growing number of third-generation sequencing related publications. Even though sparking the development of countless bioinformatics software, the streamlined nanopore data processing into standardized formats is still lacking. As an enabling step for its successful application, we developed \textit{Nanopype}, a modular and scalable pipeline. Our approach facilitates the basic steps of basecalling, alignment, methylation- and structural variant detection with exchangeable tools in each module. Optimized for the usage on high performance compute clusters, we propose a raw data management, capable of handling multiple sequencing devices placed locally and remotely. Strict version control of integrated tools and deployment as containerized software, ensure reproducibility across projects and laboratories. Finally, we analyze disease associated repeat regions utilizing targeted nanopore sequencing and the \textit{Nanopype} processing infrastructure. The expansion of unstable genomic short tandem repeats (STRs) is of particular interest as it causes more than 30 Mendelian human disorders. Long stretches of repetitive sequence render these regions inaccessible for short-read sequencing by synthesis. Furthermore, finding current nanopore basecalling algorithms insufficient to resolve the repeat length, we developed \textit{STRique}, a raw nanopore signal based repeat detection and quantification software. We demonstrate the precise analysis of repeat lengths on patient-derived samples containing C9orf72 and FMR1 repeat expansions. The additional integration of repeat- and nearby promoter-methylation levels reveal a repeat length depending gain, suggesting an epigenetic response to the expansion. Taken together, this work contributes to further increase the usability and provides novel insights based on third-generation nanopore sequencing

RLM: fast and simplified extraction of read-level methylation metrics from bisulfite sequencing data

Bisulfite sequencing data provide value beyond the straightforward methylation assessment by analyzing single-read patterns. Over the past years, various metrics have been established to explore this layer of information. However, limited compatibility with alignment tools, reference genomes or the measurements they provide present a bottleneck for most groups to routinely perform read-level analysis. To address this, we developed RLM, a fast and scalable tool for the computation of several frequently used read-level methylation statistics. RLM supports standard alignment tools, works independently of the reference genome and handles most sequencing experiment designs. RLM can process large input files with a billion reads in just a few hours on common workstations. Availability and implementation: https://github.com/sarahet/RL

Dnmt1 has de novo activity targeted to transposable elements

DNA methylation plays a critical role during development, particularly in repressing retrotransposons. The mammalian methylation landscape is dependent on the combined activities of the canonical maintenance enzyme Dnmt1 and the de novo Dnmts, 3a and 3b. Here, we demonstrate that Dnmt1 displays de novo methylation activity in vitro and in vivo with specific retrotransposon targeting. We used whole-genome bisulfite and long-read Nanopore sequencing in genetically engineered methylation-depleted mouse embryonic stem cells to provide an in-depth assessment and quantification of this activity. Utilizing additional knockout lines and molecular characterization, we show that the de novo methylation activity of Dnmt1 depends on Uhrf1, and its genomic recruitment overlaps with regions that enrich for Uhrf1, Trim28 and H3K9 trimethylation. Our data demonstrate that Dnmt1 can catalyze DNA methylation in both a de novo and maintenance context, especially at retrotransposons, where this mechanism may provide additional stability for long-term repression and epigenetic propagation throughout development

T-REX17 is a transiently expressed non-coding RNA essential for human endoderm formation

Long non-coding RNAs (lncRNAs) have emerged as fundamental regulators in various biological processes, including embryonic development and cellular differentiation. Despite much progress over the past decade, the genome-wide annotation of lncRNAs remains incomplete and many known non-coding loci are still poorly characterized. Here, we report the discovery of a previously unannotated lncRNA that is transcribed 230 kb upstream of the SOX17 gene and located within the same topologically associating domain. We termed it T-REX17 (Transcript Regulating Endoderm and activated by soX17) and show that it is induced following SOX17 activation but its expression is more tightly restricted to early definitive endoderm. Loss of T-REX17 affects crucial functions independent of SOX17 and leads to an aberrant endodermal transcriptome, signaling pathway deregulation and epithelial to mesenchymal transition defects. Consequently, cells lacking the lncRNA cannot further differentiate into more mature endodermal cell types. Taken together, our study identified and characterized T-REX17 as a transiently expressed and essential non-coding regulator in early human endoderm differentiation

Dnmt1 has de novo activity targeted to transposable elements

DNA methylation plays a critical role during development, particularly in repressing retrotransposons. The mammalian methylation landscape is dependent on the combined activities of the canonical maintenance enzyme Dnmt1 and the de novo Dnmts, 3a and 3b. Here, we demonstrate that Dnmt1 displays de novo methylation activity in vitro and in vivo with specific retrotransposon targeting. We used whole-genome bisulfite and long-read Nanopore sequencing in genetically engineered methylation-depleted mouse embryonic stem cells to provide an in-depth assessment and quantification of this activity. Utilizing additional knockout lines and molecular characterization, we show that the de novo methylation activity of Dnmt1 depends on Uhrf1, and its genomic recruitment overlaps with regions that enrich for Uhrf1, Trim28 and H3K9 trimethylation. Our data demonstrate that Dnmt1 can catalyze DNA methylation in both a de novo and maintenance context, especially at retrotransposons, where this mechanism may provide additional stability for long-term repression and epigenetic propagation throughout development.ISSN:1545-9993ISSN:1545-998