Workload measurement for molecular genetics laboratory: A survey study

Genetic testing availability in the health care system is rapidly increasing, along with the diffusion of next-generation sequencing (NGS) into diagnostics. These issues make imperative the knowledge-drive optimization of testing in the clinical setting. Time estimations of wet laboratory procedure in Italian molecular laboratories offering genetic diagnosis were evaluated to provide data suitable to adjust efficiency and optimize health policies and costs. A survey was undertaken by the Italian Society of Human Genetics (SIGU). Forty-two laboratories participated. For most molecular techniques, the most time-consuming steps are those requiring an intensive manual intervention or in which the human bias can affect the global process time-performances. For NGS, for which the study surveyed also the interpretation time, the latter represented the step that requiring longer times. We report the first survey describing the hands-on times requested for different molecular diagnostics procedures, including NGS. The analysis of this survey suggests the need of some improvements to optimize some analytical processes, such as the implementation of laboratory information management systems to minimize manual procedures in pre-analytical steps which may affect accuracy that represents the major challenge to be faced in the future setting of molecular genetics laboratory

Deconvolution of bulk blood eQTL effects into immune cell subpopulations

BACKGROUND: Expression quantitative trait loci (eQTL) studies are used to interpret the function of disease-associated genetic risk factors. To date, most eQTL analyses have been conducted in bulk tissues, such as whole blood and tissue biopsies, which are likely to mask the cell type-context of the eQTL regulatory effects. Although this context can be investigated by generating transcriptional profiles from purified cell subpopulations, current methods to do this are labor-intensive and expensive. We introduce a new method, Decon2, as a framework for estimating cell proportions using expression profiles from bulk blood samples (Decon-cell) followed by deconvolution of cell type eQTLs (Decon-eQTL). RESULTS: The estimated cell proportions from Decon-cell agree with experimental measurements across cohorts (R ≥ 0.77). Using Decon-cell, we could predict the proportions of 34 circulating cell types for 3194 samples from a population-based cohort. Next, we identified 16,362 whole-blood eQTLs and deconvoluted cell type interaction (CTi) eQTLs using the predicted cell proportions from Decon-cell. CTi eQTLs show excellent allelic directional concordance with eQTL (≥ 96-100%) and chromatin mark QTL (≥87-92%) studies that used either purified cell subpopulations or single-cell RNA-seq, outperforming the conventional interaction effect. CONCLUSIONS: Decon2 provides a method to detect cell type interaction effects from bulk blood eQTLs that is useful for pinpointing the most relevant cell type for a given complex disease. Decon2 is available as an R package and Java application (https://github.com/molgenis/systemsgenetics/tree/master/Decon2) and as a web tool (www.molgenis.org/deconvolution)

Draft Best Practice Guidelines for Molecular Analysis in Fragile X Syndrome

Participants list BP meeting Strasbourg: Jorge Paula, Porto, PortugalDraft guidelines prepared following discussions at the EMQN workshop, 23-24 November 2001, Strasbourg, France. These guidelines have been fully updated and were published in January 200

Regulation of stem cell maintenance and cell differentiation states in Arabidopsis root development

The experiments presented in this thesis topic the role of transcription factor family members in regulating growth, development, and maintenance of the Arabidopsis root. We demonstrate a conserved homeobox transcription factor regulates distal stem cell maintenance and expand the notion that the PLETHORA (PLT) family of transcription factors specifically regulates stem cell properties to a significantly broader role. In addition, we show that members of the PLT gene family can activate transcriptional targets in a dose-dependent fashion to establish a feed-forward network that regulates root growth and development. Chapter one reviews the molecular genetical approaches that have been adopted to address Arabidopsis root development. Taking embryogenesis as a starting point, we describe the genes and mechanisms involved in root meristem and stem cell patterning and maintenance. In chapter two we demonstrate a role for the homeobox gene WOX5 (WUSCHEL-RELATED 5), a homologue of WUS, in regulating distal stem cell maintenance in a non-cell autonomous fashion downstream from the SHR/SCR signaling pathway. While loss-of-function leads to stem cell differentiation, overexpression of WOX5 results in the accumulation of distal stem cells that operate independent from quiescent centre (QC) signaling. By reciprocal expression experiments we show that WOX5 and WUS are functionally equivalent in stem cell control, indicating that the organizers of both stem cell systems employ related regulators to provide stem cell maintenance signaling. Chapter three describes the functional analysis of four PLT family members that are redundantly required for embryonic root development and post-embryonic root growth in a dose-dependent fashion. Transcriptional and translational studies of the PLT proteins reveal their graded expression spanning the root meristem with highest expression in the stem cell area, intermediate levels in the transit amplifying zone, and low levels in the differentiation zone. By changing the shape of the PLT expression patterns we show that cell fate within the root meristem depends on the amount of PLT cells are subjected to; high PLT levels maintain stem cell fate, intermediate levels control cell division, and low levels allow cell elongation. Chapter four elaborates on the PLT dosage-dependent control of root cell fate by demonstrating that direct targets of PLT2 are expressed in non-overlapping expression domains within the root meristem. By manipulating PLT2 protein levels in the root meristem, we demonstrate a corresponding shift in the target expression domain; indicating PLT2 can regulate its transcriptional targets in a concentration-dependent manner. In addition, the varied identity of downstream targets suggests that PLT2 controls diversified developmental pathways associated with cell proliferation and growth. Interestingly, the identification of auxin transport facilitators, biosynthetic regulators and signaling components as direct PLT2 targets indicate a multi-level regulatory feedback into the auxin gradient that specifies root patterning. In chapter five the results presented in the previous chapters will be discussed in the light of recent insights in stem cell maintenance and specification, morphogen theory and transcriptional networks to give directions to future research

Additional file 1: Table S1. of Identification and classification of known and putative antimicrobial compounds produced by a wide variety of Bacillales species

Accession numbers of whole genome sequences (reported in Genbank) of Bacillales analyzed in this study. (XLSX 23Â kb) (XLSX 23 kb