Illuminating Choices for Library Prep: A Comparison of Library Preparation Methods for Whole Genome Sequencing of Cryptococcus neoformans Using Illumina HiSeq.

The industry of next-generation sequencing is constantly evolving, with novel library preparation methods and new sequencing machines being released by the major sequencing technology companies annually. The Illumina TruSeq v2 library preparation method was the most widely used kit and the market leader; however, it has now been discontinued, and in 2013 was replaced by the TruSeq Nano and TruSeq PCR-free methods, leaving a gap in knowledge regarding which is the most appropriate library preparation method to use. Here, we used isolates from the pathogenic fungi Cryptococcus neoformans var. grubii and sequenced them using the existing TruSeq DNA v2 kit (Illumina), along with two new kits: the TruSeq Nano DNA kit (Illumina) and the NEBNext Ultra DNA kit (New England Biolabs) to provide a comparison. Compared to the original TruSeq DNA v2 kit, both newer kits gave equivalent or better sequencing data, with increased coverage. When comparing the two newer kits, we found little difference in cost and workflow, with the NEBNext Ultra both slightly cheaper and faster than the TruSeq Nano. However, the quality of data generated using the TruSeq Nano DNA kit was superior due to higher coverage at regions of low GC content, and more SNPs identified. Researchers should therefore evaluate their resources and the type of application (and hence data quality) being considered when ultimately deciding on which library prep method to use

Dynamic Localization of Tat Protein Transport Machinery Components in Streptomyces coelicolor

The Tat pathway transports folded proteins across the bacterial cytoplasmic membrane and is a major route of protein export in the Streptomyces genus of bacteria. In this study, we have examined the localization of Tat components in the model organism Streptomyces coelicolor by constructing enhanced green fluorescent protein (eGFP) and mCherry fusions with the TatA, TatB, and TatC proteins. All three components colocalized dynamically in the vegetative hyphae, with foci of each tagged protein being prominent at the tips of emerging germ tubes and of the vegetative hyphae, suggesting that this may be a primary site of Tat secretion. Time-lapse imaging revealed that localization of the Tat components was highly dynamic during tip growth and again demonstrated a strong preference for apical sites in growing hyphae. During aerial hypha formation, TatA-eGFP and TatB-eGFP fusions relocalized to prespore compartments, indicating repositioning of Tat components during the Streptomyces life cycle

Use of an educational computer program before genetic counseling for breast cancer susceptibility: Effects on duration and content of counseling sessions

PURPOSE: Patients seeking genetic testing for inherited breast cancer risk are typically educated by genetic counselors; however, the growing demand for cancer genetic testing will likely exceed the availability of counselors trained in this area. We compared the effectiveness of counseling alone versus counseling preceded by use of a computer-based decision aid among women referred to genetic counseling for a family or personal history of breast cancer. METHODS: We developed and evaluated an interactive computer program that educates women about breast cancer, heredity, and genetic testing. Between May 2000 and September 2002, women at six study sites were randomized into either: Counselor Group (n = 105), who received standard genetic counseling, or Computer Group (n = 106), who used the interactive computer program before counseling. Clients and counselors both evaluated the effectiveness of counseling sessions, and counselors completed additional measures for the Computer Group. Counselors also recorded the duration of each session. RESULTS: Baseline characteristics did not differ significantly between groups. Participants and counselors both rated the counseling sessions as highly effective, whether or not the sessions were preceded by computer use. Computer use resulted in significantly shorter counseling sessions among women at low risk for carrying BRCA1/2 mutations. In approximately half of the sessions preceded by clients’ computer use, counselors indicated that clients’ use of the computer program affected the way they used the time, shifting the focus away from basic education toward personal risk and decision-making. CONCLUSION: This study shows that the interactive computer program “Breast Cancer Risk and Genetic Testing” is a valuable adjunct to genetic counseling. Its use before counseling can shorten counseling sessions and allow counselors to focus more on the clients’ individual risks and specific psychological concerns. As the demand for counseling services increases, a program such as this can play a valuable role in enhancing counseling efficiency

The twin-arginine translocation (Tat) protein export pathway

The twin-arginine translocation (Tat) protein export system is present in the cytoplasmic membranes of most bacteria and archaea and has the highly unusual property of transporting fully folded proteins. The system must therefore provide a transmembrane pathway that is large enough to allow the passage of structured macromolecular substrates of different sizes but that maintains the impermeability of the membrane to ions. In the Gram-negative bacterium Escherichia coli, this complex task can be achieved by using only three small membrane proteins: TatA, TatB and TatC. In this Review, we summarize recent advances in our understanding of how this remarkable machine operates. © 2012 Macmillan Publishers Limited. All rights reserved