Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

The implementation of genome sequencing in rare genetic diseases diagnosis: a pilot study from the Hong Kong genome project

SummaryBackgroundGenome sequencing (GS) has revolutionised the diagnostic odyssey of patients with rare genetic diseases (RDs) and accelerated large-scale genome projects globally. However, the impact of GS on patients with RDs is yet to be investigated among genome projects in Asia. The Hong Kong Genome Project (HKGP) was implemented to benefit patients and families with RDs in Hong Kong, and to increase the inclusiveness of Chinese genomic data. This study evaluated the impact of short read GS (srGS), complemented by long read GS (lrGS) in a subset, on individuals recruited in the pilot phase of the HKGP.MethodsGS was performed on a prospective cohort of patients with suspected genetic disease recruited by territory-wide referrals to the HKGP. All participants received srGS, while lrGS was applied to a subset to resolve technically challenging regions unclear from srGS and provide phasing information for potential compound heterozygous variants. A phenotypic-driven diagnostic workflow was implemented to filter and prioritise rare and likely disease-causing variants. The primary outcome was diagnostic yield. The impact on the diagnostic odyssey and clinical management was also assessed.FindingsA total of 1264 individuals from 520 families with a broad spectrum of RDs were recruited, with 94% of probands being Chinese. srGS was performed for all individuals and lrGS was performed in 21 individuals. The use of srGS achieved a molecular diagnosis in 24% (125/520) of probands, and an additional 4% (21/520) with the assistance from lrGS. Approximately one-third of the identified diagnostic variants being novel. Diagnostic yield was found to be significantly higher among adult probands compared to paediatric probands (32% vs 24%; p = 0.025). The diagnostic yield was significantly higher in probands without prior genetic testing (37%; n = 185) compared to those previously tested, including exome and genome sequencing (23%; n = 335) (p = 0.001). GS ended diagnostic odysseys with an average length of 15 years (0.5–59), and potentially impacted clinical management in 77% (113/146) of diagnosed probands.InterpretationThis population-based genome project shed light on the consideration of integrating srGS and lrGS in clinical workflows for RDs. The identification of unique and prevalent variants from Southeast Asia increased the inclusiveness of Chinese genomic data, contributing to greater representation and genomic diversity.FundingThe HKGP is a publicly funded genome sequencing initiative commissioned by the Health Bureau of the HKSAR Government.</p

Author Correction: Robust estimation of bacterial cell count from optical density

An amendment to this paper has been published and can be accessed via a link at the top of the paper.</jats:p

Robust estimation of bacterial cell count from optical density

AbstractOptical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals  &lt;1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data.</jats:p