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Application of next-generation sequencing using the ABI SOLiD technology to mammalian transcriptome analysis enabled a survey of the content, the complexity and the developmental dynamics of the embryonic stem cell transcriptome in the mouse. Also in this issue, Mortazavi et al . report Illumina technology–based RNA-Seq analysis of the mouse transcriptome in three different tissues. We developed a massive-scale RNA sequencing protocol, short quantitative random RNA libraries or SQRL, to survey the complexity, dynamics and sequence content of transcriptomes in a near-complete fashion. This method generates directional, random-primed, linear cDNA libraries that are optimized for next-generation short-tag sequencing. We surveyed the poly(A) + transcriptomes of undifferentiated mouse embryonic stem cells (ESCs) and embryoid bodies (EBs) at an unprecedented depth (10 Gb), using the Applied Biosystems SOLiD technology. These libraries capture the genomic landscape of expression, state-specific expression, single-nucleotide polymorphisms (SNPs), the transcriptional activity of repeat elements, and both known and new alternative splicing events. We investigated the impact of transcriptional complexity on current models of key signaling pathways controlling ESC pluripotency and differentiation, highlighting how SQRL can be used to characterize transcriptome content and dynamics in a quantitative and reproducible manner, and suggesting that our understanding of transcriptional complexity is far from complete.

Tumour cellularity, the relative proportion of tumour and normal cells in a sample, affects the sensitivity of mutation detection, copy number analysis, cancer gene expression and methylation profiling. Tumour cellularity is traditionally estimated by pathological review of sectioned specimens; however this method is both subjective and prone to error due to heterogeneity within lesions and cellularity differences between the sample viewed during pathological review and tissue used for research purposes. In this paper we describe a statistical model to estimate tumour cellularity from SNP array profiles of paired tumour and normal samples using shifts in SNP allele frequency at regions of loss of heterozygosity (LOH) in the tumour. We also provide qpure, a software implementation of the method. Our experiments showed that there is a medium correlation 0.42 ([Formula: see text]-value=0.0001) between tumor cellularity estimated by qpure and pathology review. Interestingly there is a high correlation 0.87 ([Formula: see text]-value [Formula: see text] 2.2e-16) between cellularity estimates by qpure and deep Ion Torrent sequencing of known somatic KRAS mutations; and a weaker correlation 0.32 ([Formula: see text]-value=0.004) between IonTorrent sequencing and pathology review. This suggests that qpure may be a more accurate predictor of tumour cellularity than pathology review. qpure can be downloaded from https://sourceforge.net/projects/qpure/.

Somatic mutation calling from next-generation sequencing data remains a challenge due to the difficulties of distinguishing true somatic events from artifacts arising from PCR, sequencing errors or mis-mapping. Tumor cellularity or purity, sub-clonality and copy number changes also confound the identification of true somatic events against a background of germline variants. We have developed a heuristic strategy and software (http://www.qcmg.org/bioinformatics/qsnp/) for somatic mutation calling in samples with low tumor content and we show the superior sensitivity and precision of our approach using a previously sequenced cell line, a series of tumor/normal admixtures, and 3,253 putative somatic SNVs verified on an orthogonal platform.

The development of the mammalian kidney is well conserved from mouse to man. Despite considerable temporal and spatial data on gene expression in mammalian kidney development, primarily in rodent species, there is a paucity of genes whose expression is absolutely specific to a given anatomical compartment and/or developmental stage, defined here as 'anchor' genes. We previously generated an atlas of gene expression in the developing mouse kidney using microarray analysis of anatomical compartments collected via laser capture microdissection. Here, this data is further analysed to identify anchor genes via stringent bioinformatic filtering followed by high resolution section in situ hybridisation performed on 200 transcripts selected as specific to one of 11 anatomical compartments within the midgestation mouse kidney. A total of 37 anchor genes were identified across 6 compartments with the early proximal tubule being the compartment richest in anchor genes. Analysis of minimal and evolutionarily conserved promoter regions of this set of 25 anchor genes identified enrichment of transcription factor binding sites for Hnf4a and Hnf1b, RbpJ (Notch signalling), PPARγ:RxRA and COUP-TF family transcription factors. This was reinforced by GO analyses which also identified these anchor genes as targets in processes including epithelial proliferation and proximal tubular function. As well as defining anchor genes, this large scale validation of gene expression identified a further 92 compartment-enriched genes able to subcompartmentalise key processes during murine renal organogenesis spatially or ontologically. This included a cohort of 13 ureteric epithelial genes revealing previously unappreciated compartmentalisation of the collecting duct system and a series of early tubule genes suggesting that segmentation into proximal tubule, loop of Henle and distal tubule does not occur until the onset of glomerular vascularisation. Overall, this study serves to illuminate previously ill-defined stages of patterning and will enable further refinement of the lineage relationships within mammalian kidney development.

Pancreatic cancer remains one of the most lethal of malignancies and a major health burden. We performed whole-genome sequencing and copy number variation (CNV) analysis of 100 pancreatic ductal adenocarcinomas (PDACs). Chromosomal rearrangements leading to gene disruption were prevalent, affecting genes known to be important in pancreatic cancer ( TP53, SMAD4, CDKN2A, ARID1A and ROBO2 ) and new candidate drivers of pancreatic carcinogenesis ( KDM6A and PREX2 ). Patterns of structural variation (variation in chromosomal structure) classified PDACs into 4 subtypes with potential clinical utility: the subtypes were termed stable, locally rearranged, scattered and unstable. A significant proportion harboured focal amplifications, many of which contained druggable oncogenes ( ERBB2 , MET , FGFR1 , CDK6 , PIK3R3 and PIK3CA ), but at low individual patient prevalence. Genomic instability co-segregated with inactivation of DNA maintenance genes ( BRCA1 , BRCA2 or PALB2 ) and a mutational signature of DNA damage repair deficiency. Of 8 patients who received platinum therapy, 4 of 5 individuals with these measures of defective DNA maintenance responded. A whole-genome sequencing analysis of 100 pancreatic ductal adenocarcinomas has discovered known and newly identified genetic drivers of pancreatic cancer; these genetic alterations can be classified into four subtypes, which raises the possibility of improved targeting of clinical treatments. The mutations associated with pancreatic cancer A whole-genome sequencing analysis of 100 pancreatic ductal adenocarcinomas has revealed known and newly identified genetic drivers of pancreatic carcinogenesis. These genetic alterations can be classified into four subtypes based on the patterns of structural variation — stable, locally rearranged, scattered and unstable — which raises the possibility of improved targeting of clinical treatments. A number of tumours harboured focal amplifications, many containing druggable oncogenes, although at low individual prevalence. Genomic instability co-segregated with inactivation of DNA maintenance genes and a mutational signature of DNA damage repair deficiency. In a proof-of-concept study, the authors show the potential utility of these genomic signatures as putative biomarkers for therapeutic selection.

Patients with high-grade serous ovarian cancer (HGSC) have experienced little improvement in overall survival, and standard treatment has not advanced beyond platinum-based combination chemotherapy, during the past 30 years. To understand the drivers of clinical phenotypes better, here we use whole-genome sequencing of tumour and germline DNA samples from 92 patients with primary refractory, resistant, sensitive and matched acquired resistant disease. We show that gene breakage commonly inactivates the tumour suppressors RB1 , NF1 , RAD51B and PTEN in HGSC, and contributes to acquired chemotherapy resistance. CCNE1 amplification was common in primary resistant and refractory disease. We observed several molecular events associated with acquired resistance, including multiple independent reversions of germline BRCA1 or BRCA2 mutations in individual patients, loss of BRCA1 promoter methylation, an alteration in molecular subtype, and recurrent promoter fusion associated with overexpression of the drug efflux pump MDR1. Whole-genome sequencing of tumour and germline DNA samples from 92 patients with high-grade serous ovarian cancer identifies frequent gene breakages that inactivate the tumour suppressors RB1, NF1, RAD51B and PTEN , and contribute to chemotherapy resistance; acquired resistance was associated with diverse mechanisms such as reversions of germline BRCA1/2 mutations and overexpression of the drug efflux pump MDR1. Drug-resistant ovarian cancers sequenced Although responsive to initial chemotherapy, high-grade serous ovarian cancer (HGSC) frequently develops resistance. David Bowtell and colleagues performed whole-genome sequencing of tumour and germline DNA samples from 92 HGSC patients. Their analyses identify frequent gene breakage that inactivates tumour suppressors RB1 , NF1 , RAD51B and PTEN , contributing to acquired chemotherapy resistance. Primary resistant and refractory disease was associated with CCNE1 amplification. Acquired resistance was associated with diverse mechanisms such as reversions of germline BRCA1 or BRCA2 mutations, loss of BRCA1 promoter methylation, an alteration in molecular subtype, and overexpression of the drug efflux pump MDR1.

Research Question: What are the project performance differences between Irish construction firms that have adopted Lean thinking and practices and those that have not? Purpose: This study explores Percent Plan Complete (PPC) as part of the application of Last Planner® System (LPS) across two concurrent capital projects in Ireland. It examines commonalities and differences between trade contractors' PPC, and it identifies areas of improvement for implementation on future projects. Research Method: Mixed-methods approach encompassing critical literature review, site documentation data-analysis, focus groups, and semi-structured purposeful interviews. Findings: Lean Construction-oriented contractor selection, early engagement of trades in the design process, implementation of all functions of LPS, education and training in Lean, increased modularization and prefabrication, and embracing technological advances are posited as areas for focused improvement. Limitations: This study is limited to examination of PPC data from two concurrent Engineering, Procurement, Construction Management, Validation (EPCMV) Projects. Implications: Irish construction firms should adopt Lean thinking and practices to provide added value on capital projects. There is a need for further studies on the PPC performances and reasons for non-completion (RNC) of trade contractors across multiple projects.

Purpose The purpose of this paper is to extend the People Value Stream concept further by developing a view of what the world would look like through the eyes of a positive psychology employee-centred lens. The authors hope to provide a frame for further discussion, research and practical application in this area. Design/methodology/approach In this conceptual paper, the authors draw on their collective 120 plus years of experience with Lean and Human Resource Management through leading, teaching, researching and consulting in the area. Findings The People Value Stream concept is extended here by ideating how the “Voice of the Employee” could be used to enhance the existing knowledge of Lean. Relying on a range of cognitive psychological theories, particularly Self-Determination Theory, the authors show how it might be possible to develop a highly engaged workforce primarily by unlocking their intrinsic motivation through a “Self-Development and Growth Cycle”. This cycle is the people-improvement version of the seminal Deming process-improvement PDCA cycle. It can be applied within a job crafting “Personal Cockpit”. The authors also highlight a range of outputs and wider implications that create a pull for team leaders and senior management wishing to move to a real Servant Leader model. It will also help those developing and supporting people-related policies and procedures both within organisations and in trade unions. Originality/value This paper turns the existing literature about people within Lean upside down. To the best of the authors’ knowledge, for the first time in an academic paper, it discusses what would be the implications for the Lean world if the authors truly started understanding and deploying the explicit “Voice of the Employee” rather than just the established Lean “Voice of the Owner”-led Hoshin Kanri approach. The authors show how a lack of knowledge in these areas by the Lean community is limiting Lean’s engagement of people and its sustainability.

ObjectiveExtensive molecular heterogeneity of pancreatic ductal adenocarcinoma (PDA), few effective therapies and high mortality make this disease a prime model for advancing development of tailored therapies. The p16-cyclin D-cyclin-dependent kinase 4/6-retinoblastoma (RB) protein (CDK4) pathway, regulator of cell proliferation, is deregulated in PDA. Our aim was to develop a novel personalised treatment strategy for PDA based on targeting CDK4.DesignSensitivity to potent CDK4/6 inhibitor PD-0332991 (palbociclib) was correlated to protein and genomic data in 19 primary patient-derived PDA lines to identify biomarkers of response. In vivo efficacy of PD-0332991 and combination therapies was determined in subcutaneous, intrasplenic and orthotopic tumour models derived from genome-sequenced patient specimens and genetically engineered model. Mechanistically, monotherapy and combination therapy were investigated in the context of tumour cell and extracellular matrix (ECM) signalling. Prognostic relevance of companion biomarker, RB protein, was evaluated and validated in independent PDA patient cohorts (>500 specimens).ResultsSubtype-specific in vivo efficacy of PD-0332991-based therapy was for the first time observed at multiple stages of PDA progression: primary tumour growth, recurrence (second-line therapy) and metastatic setting and may potentially be guided by a simple biomarker (RB protein). PD-0332991 significantly disrupted surrounding ECM organisation, leading to increased quiescence, apoptosis, improved chemosensitivity, decreased invasion, metastatic spread and PDA progression in vivo. RB protein is prevalent in primary operable and metastatic PDA and may present a promising predictive biomarker to guide this therapeutic approach.ConclusionThis study demonstrates the promise of CDK4 inhibition in PDA over standard therapy when applied in a molecular subtype-specific context.

Pancreatic cancer is a highly lethal malignancy with few effective therapies. We performed exome sequencing and copy number analysis to define genomic aberrations in a prospectively accrued clinical cohort ( n = 142) of early (stage I and II) sporadic pancreatic ductal adenocarcinoma. Detailed analysis of 99 informative tumours identified substantial heterogeneity with 2,016 non-silent mutations and 1,628 copy-number variations. We define 16 significantly mutated genes, reaffirming known mutations ( KRAS , TP53 , CDKN2A, SMAD4 , MLL3 , TGFBR2, ARID1A and SF3B1 ), and uncover novel mutated genes including additional genes involved in chromatin modification ( EPC1 and ARID2 ), DNA damage repair ( ATM ) and other mechanisms ( ZIM2 , MAP2K4 , NALCN , SLC16A4 and MAGEA6 ). Integrative analysis with in vitro functional data and animal models provided supportive evidence for potential roles for these genetic aberrations in carcinogenesis. Pathway-based analysis of recurrently mutated genes recapitulated clustering in core signalling pathways in pancreatic ductal adenocarcinoma, and identified new mutated genes in each pathway. We also identified frequent and diverse somatic aberrations in genes described traditionally as embryonic regulators of axon guidance, particularly SLIT/ROBO signalling, which was also evident in murine Sleeping Beauty transposon-mediated somatic mutagenesis models of pancreatic cancer, providing further supportive evidence for the potential involvement of axon guidance genes in pancreatic carcinogenesis. Exome sequencing and copy number analysis are used to define genomic aberrations in early sporadic pancreatic ductal adenocarcinoma; among the findings are mutations in genes involved in chromatin modification and DNA damage repair, and frequent and diverse somatic aberrations in genes known as embryonic regulators of axon guidance. New mutations identified in pancreatic cancer This large-scale study presents exome sequencing and copy number variant analysis from 142 patients with pancreatic ductal adenocarcinoma, the most common form of pancreatic cancer. Among the findings are mutations in genes involved in chromatin modification and DNA damage repair, not previously implicated in this disease. Importantly, the data show that abnormal expression of genes involved in slit and semaphorin signalling is associated with poor patient survival, and in animal models was associated with disease development and progression.