A comprehensive assessment of somatic mutation detection in cancer using whole-genome sequencing.

As whole-genome sequencing for cancer genome analysis becomes a clinical tool, a full understanding of the variables affecting sequencing analysis output is required. Here using tumour-normal sample pairs from two different types of cancer, chronic lymphocytic leukaemia and medulloblastoma, we conduct a benchmarking exercise within the context of the International Cancer Genome Consortium. We compare sequencing methods, analysis pipelines and validation methods. We show that using PCR-free methods and increasing sequencing depth to ∼ 100 × shows benefits, as long as the tumour:control coverage ratio remains balanced. We observe widely varying mutation call rates and low concordance among analysis pipelines, reflecting the artefact-prone nature of the raw data and lack of standards for dealing with the artefacts. However, we show that, using the benchmark mutation set we have created, many issues are in fact easy to remedy and have an immediate positive impact on mutation detection accuracy.We thank the DKFZ Genomics and Proteomics Core Facility and the OICR Genome Technologies Platform for provision of sequencing services. Financial support was provided by the consortium projects READNA under grant agreement FP7 Health-F4-2008-201418, ESGI under grant agreement 262055, GEUVADIS under grant agreement 261123 of the European Commission Framework Programme 7, ICGC-CLL through the Spanish Ministry of Science and Innovation (MICINN), the Instituto de Salud Carlos III (ISCIII) and the Generalitat de Catalunya. Additional financial support was provided by the PedBrain Tumor Project contributing to the International Cancer Genome Consortium, funded by German Cancer Aid (109252) and by the German Federal Ministry of Education and Research (BMBF, grants #01KU1201A, MedSys #0315416C and NGFNplus #01GS0883; the Ontario Institute for Cancer Research to PCB and JDM through funding provided by the Government of Ontario, Ministry of Research and Innovation; Genome Canada; the Canada Foundation for Innovation and Prostate Cancer Canada with funding from the Movember Foundation (PCB). PCB was also supported by a Terry Fox Research Institute New Investigator Award, a CIHR New Investigator Award and a Genome Canada Large-Scale Applied Project Contract. The Synergie Lyon Cancer platform has received support from the French National Institute of Cancer (INCa) and from the ABS4NGS ANR project (ANR-11-BINF-0001-06). The ICGC RIKEN study was supported partially by RIKEN President’s Fund 2011, and the supercomputing resource for the RIKEN study was provided by the Human Genome Center, University of Tokyo. MDE, LB, AGL and CLA were supported by Cancer Research UK, the University of Cambridge and Hutchison-Whampoa Limited. SD is supported by the Torres Quevedo subprogram (MI CINN) under grant agreement PTQ-12-05391. EH is supported by the Research Council of Norway under grant agreements 221580 and 218241 and by the Norwegian Cancer Society under grant agreement 71220-PR-2006-0433. Very special thanks go to Jennifer Jennings for administrating the activity of the ICGC Verification Working Group and Anna Borrell for administrative support.This is the final version of the article. It first appeared from Nature Publishing Group via http://dx.doi.org/10.1038/ncomms1000

Time-Lapse Imaging of Neuroblastoma Cells to Determine Cell Fate upon Gene Knockdown

<div><p>Neuroblastoma is the most common extra-cranial solid tumor of early childhood. Standard therapies are not effective in case of poor prognosis and chemotherapy resistance. To improve drug therapy, it is imperative to discover new targets that play a substantial role in tumorigenesis of neuroblastoma. The mitotic machinery is an attractive target for therapeutic interventions and inhibitors can be developed to target mitotic entry, spindle apparatus, spindle activation checkpoint, and mitotic exit. We present an elaborate analysis pipeline to determine cancer specific therapeutic targets by first performing a focused gene expression analysis to select genes followed by a gene knockdown screening assay of live cells. We interrogated gene expression studies of neuroblastoma tumors and selected 240 genes relevant for tumorigenesis and cell cycle. With these genes we performed time-lapse screening of gene knockdowns in neuroblastoma cells. We classified cellular phenotypes and used the temporal context of the perturbation effect to determine the sequence of events, particularly the mitotic entry preceding cell death. Based upon this phenotype kinetics from the gene knockdown screening, we inferred dynamic gene functions in mitosis and cell proliferation. We identified six genes (<em>DLGAP5</em>, <em>DSCC1</em>, <em>SMO</em>, <em>SNRPD1</em>, <em>SSBP1</em>, and <em>UBE2C</em>) with a vital role in mitosis and these are promising therapeutic targets for neuroblastoma. Images and movies of every time point of all screened genes are available at <a href="https://ichip.bioquant.uni-heidelberg.de">https://ichip.bioquant.uni-heidelberg.de</a>.</p> </div

Consequences of a gene knockdown on the cell cycle and cell fate.

<p>These effects can be observed (directly or indirectly) by imaging cells with silenced genes following a mitotic time-lapse screening assay. Cells may directly be affected from a loss-of-function of a gene and die (cell death), they may enter mitosis and die before completion of mitosis (cell death in mitotic arrest) or may undergo mitotic slippage followed by interphase arrest or cell death <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050988#pone.0050988-Manchado1" target="_blank">[23]</a>.</p

Determination of candidate genes that show cell death during or after mitosis.

<p>The population response to a knockdown was computed to identify the sequence of phenotype occurrence.</p

The workflow.

<p>(A) Neuroblastoma associated genes were selected based on gene expression profiles of neuroblastoma tumors and cell lines, (B) selected genes were subjected to image-based time-lapse siRNA knockdown screens, (C) each cell in an image was classified into one of the phenotype classes interphase, mitosis, or cell death, and (D) time series of the phenotypes were assembled into phenotype profiles to determine gene function of each gene knockdown.</p

Confusion matrix of the classification results after automated correction.

<p>Confusion matrix of the classification results after automated correction.</p

Time series of interphase cells during five days of screening.

<p>The population shows a periodicity of ∼35 hours representing the cell cycle duration (blue bars: interphase counts (normalized by B-Score normalization) of all screened cells for each time-frame, red curve: fitting curve).</p

Prenatal maternal stress and wheeze in children:Novel insights into epigenetic regulation

Psychological stress during pregnancy increases the risk of childhood wheeze and asthma. However, the transmitting mechanisms remain largely unknown. Since epigenetic alterations have emerged as a link between perturbations in the prenatal environment and an increased disease risk we used whole genome bisulfite sequencing (WGBS) to analyze changes in DNA methylation in mothers and their children related to prenatal psychosocial stress and assessed its role in the development of wheeze in the child. We evaluated genomic regions altered in their methylation level due to maternal stress based of WGBS data of 10 mother-child-pairs. These data were complemented by longitudinal targeted methylation and transcriptional analyses in children from our prospective mother-child cohort LINA for whom maternal stress and wheezing information was available (n = 443). High maternal stress was associated with an increased risk for persistent wheezing in the child until the age of 5. Both mothers and children showed genome-wide alterations in DNA-methylation specifically in enhancer elements. Deregulated neuroendocrine and neurotransmitter receptor interactions were observed in stressed mothers and their children. In children but not in mothers, calcium- and Wnt-signaling required for lung maturation in the prenatal period were epigenetically deregulated and could be linked with wheezing later in children’s life