Benchmarking of 4C-seq pipelines based on real and simulated data.

MOTIVATION: With its capacity for high-resolution data output in one region of interest, chromosome conformation capture combined with high-throughput sequencing (4C-seq) is a state-of-the-art next-generation sequencing technique that provides epigenetic insights, and regularly advances current medical research. However, 4C-seq data are complex and prone to biases, and while specialized programs exist, an unbiased, extensive benchmarking is still lacking. Furthermore, neither substantial datasets with fully characterized ground truth, nor simulation programs for realistic 4C-seq data have been published. RESULTS: We conducted a benchmarking study on 66 4C-seq samples from 20 datasets, and developed a novel 4C-seq simulation software, Basic4CSim, to allow for detailed comparisons of 4C-seq algorithms on 50 simulated datasets with 10-120 samples each. Simulations and benchmarking were adapted to address different characteristics of 4C-seq data. Simulated data were compared with published samples to validate simulation settings. We identified differences between 4C-seq algorithms in terms of precision, recall, interaction structure, and run time, and observed general trends. Novel differential pipeline versions of single-sample based 4C-seq algorithms were included in the benchmarking. While no single tool was optimally suited for both near-cis and far-cis, and both single-sample and differential analyses, choosing a high-performing algorithm variant did improve results considerably. For near-cis scenarios, r3Cseq, peakC and FourCSeq offered high precision, while fourSig demonstrated high overall F1 scores in far-cis analyses. Finally, 4C-seq simulations may aid in the development of improved analysis algorithms. AVAILABILITY AND IMPLEMENTATION: Basic4CSim is available at https://github.com/walter-ca/Basic4CSim. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Temporal autoregulation during human PU.1 locus SubTAD formation.

Epigenetic control of gene expression occurs within discrete spatial chromosomal units called topologically associating domains (TADs), but the exact spatial requirements of most genes are unknown; this is of particular interest for genes involved in cancer. We therefore applied high-resolution chromosomal conformation capture sequencing to map the three-dimensional (3D) organization of the human locus encoding the key myeloid transcription factor PU.1 in healthy monocytes and acute myeloid leukemia (AML) cells. We identified a dynamic ∼75-kb unit (SubTAD) as the genomic region in which spatial interactions between PU.1 gene regulatory elements occur during myeloid differentiation and are interrupted in AML. Within this SubTAD, proper initiation of the spatial chromosomal interactions requires PU.1 autoregulation and recruitment of the chromatin-adaptor protein LDB1 (LIM domain-binding protein 1). However, once these spatial interactions have occurred, LDB1 stabilizes them independently of PU.1 autoregulation. Thus, our data support that PU.1 autoregulates its expression in a "hit-and-run" manner by initiating stable chromosomal loops that result in a transcriptionally active chromatin architecture.

Basic4Cseq: an R/Bioconductor package for analyzing 4C-seq data.

SUMMARY: Basic4Cseq is an R/Bioconductor package for basic filtering, analysis and subsequent near-cis visualization of 4C-seq data. The package processes aligned 4C-seq raw data stored in binary alignment/map (BAM) format and maps the short reads to a corresponding virtual fragment library. Functions are included to create virtual fragment libraries providing chromosome position and further information on 4C-seq fragments (length and uniqueness of the fragment ends, and blindness of a fragment) for any BSGenome package. An optional filter is included for BAM files to remove invalid 4C-seq reads, and further filter functions are offered for 4C-seq fragments. Additionally, basic quality controls based on the read distribution are included. Fragment data in the vicinity of the experiment's viewpoint are visualized as coverage plot based on a running median approach and a multi-scale contact profile. Wig files or csv files of the fragment data can be exported for further analyses and visualizations of interactions with other programs. AVAILABILITY AND IMPLEMENTATION: Basic4Cseq is implemented in R and available at http://www.bioconductor.org/. A vignette with detailed descriptions of the functions is included in the package. CONTACT: Carolin.Walter@uni-muenster.de SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Cancer therapy monitoring in xenografts by quantitative analysis of circulating tumor DNA.

CONTEXT: Circulating tumor DNA (ctDNA) is a promising biomarker in cancer. MATERIALS AND METHODS: We generated xenograft models of cancer and detected ctDNA in plasma by qRCR targeting human AluJ sequences. RESULTS: Our assay reached single cell sensitivity in vitro and a correlation between ctDNA amount and tumor size was observed in vivo. Treatment with a mitogen activated protein kinase kinase (MEK)-inhibitor (BAY 869766) reduced ctDNA levels. Using this assay, we also confirmed that high levels of cell-free DNA are found in cancer patients compared to healthy individuals. DISCUSSION AND CONCLUSION: We show that ctDNA may be useful biomarker for monitoring tumor growth and treatment response.

Label-free enrichment of avian Leucocytozoon using flow cytometric sorting.

The group of haemosporidian parasites is of general interest to basic and applied science, since several species infect mammals, leading to malaria and associated disease symptoms. Although the great majority of haemosporidian parasites appear in bird hosts, as in the case of Leucocytozoon buteonis, there is little genomic information about genetic aspects of their co-evolution with hosts. Consequently, there is a high need for parasite-enrichment strategies enabling further analyses of the genomes, namely without exposure to DNA-intercalating dyes. Here, we used flow cytometry without an additional labelling step to enrich L. buteonis from infected buzzard blood. A specific, defined area of two-dimensional scattergramms was sorted and the fraction was further analysed. The successful enrichment of L. buteonis in the sorted fraction was demonstrated by Giemsa-staining and qPCR revealing a clear increase of parasite-specific genes, while host-specific genes were significantly decreased. This is the first report describing a labelling-free enrichment approach of L. buteonis from infected buzzard blood. The enrichment of parasites presented here is free of nucleic acid-intercalating dyes which may interfere with fluorescence-based methods or subsequent sequencing approaches.