Philipp Rescheneder

NextGenMap: fast and accurate read mapping in highly polymorphic genomes

SUMMARY When choosing a read mapper, one faces the trade off between speed and the ability to map reads in highly polymorphic regions. Here, we report NextGenMap, a fast and accurate read mapper, which reduces this dilemma. NextGenMap aligns reads reliably to a reference genome even when the sequence difference between target and reference genome is large, i.e. highly polymorphic genome. At the same time, NextGenMap outperforms current mapping methods with respect to runtime and to the number of correctly mapped reads. NextGenMap efficiently uses the available hardware by exploiting multi-core CPUs as well as graphic cards (GPUs), if available. In addition, NextGenMap handles automatically any read data independent of read length and sequencing technology. AVAILABILITY NextGenMap source code and documentation are available at: http://cibiv.github.io/NextGenMap/. CONTACT fritz.sedlazeck@univie.ac.at. SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

A-type lamins bind both hetero- and euchromatin, the latter being regulated by lamina-associated polypeptide 2 alpha.

Lamins are components of the peripheral nuclear lamina and interact with heterochromatic genomic regions, termed lamina-associated domains (LADs). In contrast to lamin B1 being primarily present at the nuclear periphery, lamin A/C also localizes throughout the nucleus, where it associates with the chromatin-binding protein lamina-associated polypeptide (LAP) 2 alpha. Here, we show that lamin A/C also interacts with euchromatin, as determined by chromatin immunoprecipitation of euchromatin- and heterochromatin-enriched samples. By way of contrast, lamin B1 was only found associated with heterochromatin. Euchromatic regions occupied by lamin A/C overlap with those bound by LAP2alpha, and lack of LAP2alpha in LAP2alpha-deficient cells shifts binding of lamin A/C toward more heterochromatic regions. These alterations in lamin A/C-chromatin interactions correlate with changes in epigenetic histone marks in euchromatin but do not significantly affect gene expression. Loss of lamin A/C in heterochromatic regions in LAP2alpha-deficient cells, however, correlated with increased gene expression. Our data show a novel role of nucleoplasmic lamin A/C and LAP2alpha in regulating euchromatin.

Revisiting the coding potential of the E. coli genome through Hfq co-immunoprecipitation

Hfq is a global regulator of gene expression in bacteria undergoing adaptation to changing environmental conditions. Its major function is to promote RNA-RNA interactions between regulatory small RNAs (sRNAs) and their target mRNAs. Previously, we demonstrated that Hfq binds many antisense RNAs (asRNAs) in vitro and hypothesized that Hfq may play a role in regulating gene expression via asRNAs. To investigate the E. coli Hfq-binding transcriptome in more detail, we co-immunoprecipitated and deep-sequenced RNAs bound to Hfq in vivo. We detected many new Hfq-binding sRNAs and observed that almost 300 mRNAs bind to Hfq. Among these, several are known to be sRNA targets. We identified 25 novel RNAs, which are transcribed from within protein coding regions and named them intragenic RNAs (intraRNAs). Furthermore, 67 asRNAs were co-immunoprecipitated with Hfq, demonstrating that Hfq binds antisense transcripts in vivo. Northern blot analyses confirmed the deep sequencing results and demonstrated that many of the novel Hfq-binding RNAs identified are regulated by Hfq.

The Borrelia burgdorferi RelA/SpoT Homolog and Stringent Response Regulate Survival in the Tick Vector and Global Gene Expression during Starvation

As the Lyme disease bacterium Borrelia burgdorferi traverses its enzootic cycle, alternating between a tick vector and a vertebrate host, the spirochete must adapt and persist in the tick midgut under prolonged nutrient stress between blood meals. In this study, we examined the role of the stringent response in tick persistence and in regulation of gene expression during nutrient limitation. Nutritionally starving B. burgdorferi in vitro increased the levels of guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), collectively referred to as (p)ppGpp, products of the bifunctional synthetase/hydrolase RelBbu (RelA/SpoT homolog). Conversely, returning B. burgdorferi to a nutrient-rich medium decreased (p)ppGpp levels. B. burgdorferi survival in ticks between the larval and nymph blood meals, and during starvation in vitro, was dependent on RelBbu. Furthermore, normal morphological conversion from a flat-wave shape to a condensed round body (RB) form during starvation was dependent on RelBbu; rel Bbu mutants more frequently formed RBs, but their membranes were compromised. By differential RNA sequencing analyses, we found that RelBbu regulates an extensive transcriptome, both dependent and independent of nutrient stress. The RelBbu regulon includes the glp operon, which is important for glycerol utilization and persistence in the tick, virulence factors and the late phage operon of the 32-kb circular plasmid (cp32) family. In summary, our data suggest that RelBbu globally modulates transcription in response to nutrient stress by increasing (p)ppGpp levels to facilitate B. burgdorferi persistence in the tick.

Accurate detection of complex structural variations using single-molecule sequencing

Structural variations are the greatest source of genetic variation, but they remain poorly understood because of technological limitations. Single-molecule long-read sequencing has the potential to dramatically advance the field, although high error rates are a challenge with existing methods. Addressing this need, we introduce open-source methods for long-read alignment (NGMLR; https://github.com/philres/ngmlr) and structural variant identification (Sniffles; https://github.com/fritzsedlazeck/Sniffles) that provide unprecedented sensitivity and precision for variant detection, even in repeat-rich regions and for complex nested events that can have substantial effects on human health. In several long-read datasets, including healthy and cancerous human genomes, we discovered thousands of novel variants and categorized systematic errors in short-read approaches. NGMLR and Sniffles can automatically filter false events and operate on low-coverage data, thereby reducing the high costs that have hindered the application of long reads in clinical and research settings.NGMLR and Sniffles perform highly accurate alignment and structural variation detection from long-read sequencing data.

Teaser: Individualized benchmarking and optimization of read mapping results for NGS data

Mapping reads to a genome remains challenging, especially for non-model organisms with lower quality assemblies, or for organisms with higher mutation rates. While most research has focused on speeding up the mapping process, little attention has been paid to optimize the choice of mapper and parameters for a user’s dataset. Here, we present Teaser, a software that assists in these choices through rapid automated benchmarking of different mappers and parameter settings for individualized data. Within minutes, Teaser completes a quantitative evaluation of an ensemble of mapping algorithms and parameters. We use Teaser to demonstrate how Bowtie2 can be optimized for different data.

Thiol-linked alkylation of RNA to assess expression dynamics

Gene expression profiling by high-throughput sequencing reveals qualitative and quantitative changes in RNA species at steady state but obscures the intracellular dynamics of RNA transcription, processing and decay. We developed thiol(SH)-linked alkylation for the metabolic sequencing of RNA (SLAM seq), an orthogonal-chemistry-based RNA sequencing technology that detects 4-thiouridine (s4U) incorporation in RNA species at single-nucleotide resolution. In combination with well-established metabolic RNA labeling protocols and coupled to standard, low-input, high-throughput RNA sequencing methods, SLAM seq enabled rapid access to RNA-polymerase-II-dependent gene expression dynamics in the context of total RNA. We validated the method in mouse embryonic stem cells by showing that the RNA-polymerase-II-dependent transcriptional output scaled with Oct4/Sox2/Nanog-defined enhancer activity, and we provide quantitative and mechanistic evidence for transcript-specific RNA turnover mediated by post-transcriptional gene regulatory pathways initiated by microRNAs and N6-methyladenosine. SLAM seq facilitates the dissection of fundamental mechanisms that control gene expression in an accessible, cost-effective and scalable manner.

SLAM-seq defines direct gene-regulatory functions of the BRD4-MYC axis

Profiling transcription—a SLAM dunk Identification of the direct target genes of transcription factors could shed light on how healthy cells become malignant. Muhar et al. applied a modified version of a transcript-mapping method called SLAM-seq to identify the target genes of two transcriptional regulators of major interest in cancer research (see the Perspective by Sabò and Amati). The MYC oncoprotein selectively activates transcription of just a few genes, primarily those involved in basic cell metabolism. In contrast, BRD4, a bromodomain-containing protein that is being targeted for cancer therapy, activates transcription of many genes. Science, this issue p. 800; see also p. 713 An mRNA mapping method identifies the direct gene targets of two transcriptional regulators implicated in cancer. Defining direct targets of transcription factors and regulatory pathways is key to understanding their roles in physiology and disease. We combined SLAM-seq [thiol(SH)–linked alkylation for the metabolic sequencing of RNA], a method for direct quantification of newly synthesized messenger RNAs (mRNAs), with pharmacological and chemical-genetic perturbation in order to define regulatory functions of two transcriptional hubs in cancer, BRD4 and MYC, and to interrogate direct responses to BET bromodomain inhibitors (BETis). We found that BRD4 acts as general coactivator of RNA polymerase II–dependent transcription, which is broadly repressed upon high-dose BETi treatment. At doses triggering selective effects in leukemia, BETis deregulate a small set of hypersensitive targets including MYC. In contrast to BRD4, MYC primarily acts as a selective transcriptional activator controlling metabolic processes such as ribosome biogenesis and de novo purine synthesis. Our study establishes a simple and scalable strategy to identify direct transcriptional targets of any gene or pathway.

Complex rearrangements and oncogene amplifications revealed by long-read DNA and RNA sequencing of a breast cancer cell line

The SK-BR-3 cell line is one of the most important models for HER2+ breast cancers, which affect one in five breast cancer patients. SK-BR-3 is known to be highly rearranged, although much of the variation is in complex and repetitive regions that may be underreported. Addressing this, we sequenced SK-BR-3 using long-read single molecule sequencing from Pacific Biosciences and develop one of the most detailed maps of structural variations (SVs) in a cancer genome available, with nearly 20,000 variants present, most of which were missed by short-read sequencing. Surrounding the important ERBB2 oncogene (also known as HER2), we discover a complex sequence of nested duplications and translocations, suggesting a punctuated progression. Full-length transcriptome sequencing further revealed several novel gene fusions within the nested genomic variants. Combining long-read genome and transcriptome sequencing enables an in-depth analysis of how SVs disrupt the genome and sheds new light on the complex mechanisms involved in cancer genome evolution.

The Stringent Response-Regulated sRNA Transcriptome of Borrelia burgdorferi

The Lyme disease spirochete Borrelia (Borreliella) burgdorferi must tolerate nutrient stress to persist in the tick phase of its enzootic life cycle. We previously found that the stringent response mediated by RelBbu globally regulates gene expression to facilitate persistence in the tick vector. Here, we show that RelBbu regulates the expression of a swath of small RNAs (sRNA), affecting 36% of previously identified sRNAs in B. burgdorferi. This is the first sRNA regulatory mechanism identified in any spirochete. Threefold more sRNAs were RelBbu-upregulated than downregulated during nutrient stress and included antisense, intergenic and 5′ untranslated region sRNAs. RelBbu-regulated sRNAs associated with genes known to be important for host infection (bosR and dhhp) as well as persistence in the tick (glpF and hk1) were identified, suggesting potential mechanisms for post-transcriptional regulation of gene expression.