A43 Translational research: NGS metagenomics into clinical diagnostics

Abstract As research next-generation sequencing (NGS) metagenomic pipelines transition to clinical diagnostics, the user-base changes from bioinformaticians to biologists, medical doctors, and lab-technicians. Besides the obvious need for benchmarking and assessment of diagnostic outcomes of the pipelines and tools, other focus points remain: reproducibility, data immutability, user-friendliness, portability/scalability, privacy, and a clear audit trail. We have a research metagenomics pipeline that takes raw fastq files and produces annotated contigs, but it is too complicated for non-bioinformaticians. Here, we present preliminary findings in adapting this pipeline for clinical diagnostics. We used information available on relevant fora (www.bioinfo-core.org) and experiences and publications from colleague bioinformaticians in other institutes (COMPARE, UBC, and LUMC). From this information, a robust and user-friendly storage and analysis workflow was designed for non-bioinformaticians in a clinical setting. Via Conda [https://conda.io] and Docker containers [http://www.docker.com], we made our disparate pipeline processes self-contained and reproducible. Furthermore, we moved all pipeline settings into a separate JSON file. After every analysis, the pipeline settings and virtual-environment recipes will be archived (immutably) under a persistent unique identifier. This allows long-term precise reproducibility. Likewise, after every run the raw data and final products will be automatically archived, complying with data retention laws/guidelines. All the disparate processes in the pipeline are parallelized and automated via Snakemake1 (i.e. end-users need no coding skills). In addition, interactive web-reports such as MultiQC [http://multiqc.info] and Krona2 are generated automatically. By combining Snakemake, Conda, and containers, our pipeline is highly portable and easily scaled up for outbreak situations, or scaled down to reduce costs. Since patient privacy is a concern, our pipeline automatically removes human genetic data. Moreover, all source code will be stored on an internal Gitlab server, and, combined with the archived data, ensures a clear audit trail. Nevertheless, challenges remain: (1) reproducible reference databases, e.g. being able to revert to an older version to reproduce old analyses. (2) A user-friendly GUI. (3) Connecting the pipeline and NGS data to in-house LIMS. (4) Efficient long-term storage, e.g. lossless compression algorithms. Nevertheless, this work represents a step forward in making user-friendly clinical diagnostic workflows.

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Miniaturization of analytical systems

Clinical Chemistry ◽

10.1093/clinchem/44.9.2008 ◽

1998 ◽

Vol 44 (9) ◽

pp. 2008-2014 ◽

Cited By ~ 22

Author(s):

Larry J Kricka

Keyword(s):

Self Assembly ◽

New Technologies ◽

Analytical Techniques ◽

Sample Volume ◽

Clinical Diagnostics ◽

Key Issues ◽

User Friendly ◽

Near Future ◽

Long Term Trend

Abstract Miniaturization has been a long-term trend in clinical diagnostics instrumentation. Now a range of new technologies, including micromachining and molecular self-assembly, are providing the means for further size reduction of analyzers to devices with micro- to nanometer dimensions and submicroliter volumes. Many analytical techniques (e.g., mass spectrometry and electrophoresis) have been successfully implemented on microchips made from silicon, glass, or plastic. The new impetus for miniaturization stems from the perceived benefits of faster, easier, less costly, and more convenient analyses and by the needs of the pharmaceutical industry for microscale, massively parallel drug discovery assays. Perfecting a user-friendly interface between a human and a microchip and determining the realistic lower limit for sample volume are key issues in the future implementation of these devices. Resolution of these issues will be important for the long-term success of microminiature analyzers; in the meantime, the scope, diversity, and rate of progress in the development of these devices promises products in the near future.

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Development of a fast and user-friendly cryopreservation protocol for sweet potato genetic resources

Scientific Reports ◽

10.1038/s41598-020-70869-3 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Hannes Wilms ◽

Natalia Fanega Sleziak ◽

Maarten Van der Auweraer ◽

Martijn Brands ◽

Matthijs Verleije ◽

...

Keyword(s):

Sweet Potato ◽

Ipomoea Batatas ◽

Solution Treatment ◽

Regeneration Rate ◽

Droplet Vitrification ◽

Long Term Storage ◽

User Friendly ◽

Cryopreservation Protocol ◽

Term Storage

Abstract Sweet potato (Ipomoea batatas) is one of the ten most important staple crops and provides a livelihood for many people around the globe. To adapt to ever-changing circumstances farmers and breeders need to have access to a broad diversity of germplasm. This study focuses on the development of a cryopreservation protocol that allows the long term storage of different sweet potato cultivars. For this, a droplet vitrification protocol was optimized, comparing several parameters; preculture method (0.3 M sucrose vs no preculture); meristem position (axillary vs apical); plant age (3 to 9 weeks); regeneration medium (MS + 2.22 µM BA, Hirai and MS); and length of loading solution treatment (20 to 360 min). Two months after cryopreservation, the regeneration rates of the meristems were compared, which resulted in significant differences for the preculture method, meristem position and loading solution. With these new insights an optimized droplet vitrification protocol was developed with the following parameters: use of 3–9 week old axillary meristems, no preculture phase, 20 min LS treatment, 30 min PVS2 treatment, exposure to liquid nitrogen by droplet vitrification, warming treatment in RS for 15 min, 1 day 0.3 M sucrose recuperation culture, 1 month MS + 2.22 µM BA followed by 1 month of MS cultures. This protocol was subsequently tested on 10 representative accessions resulting in a post cryopreservation regeneration rate of more than 40% for 70% of the tested cultivars, showing that this protocol could be implemented for a large portion of existing sweet potato collections.

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Evaluation of Storage Tubes for Combined Analysis of Circulating Nucleic Acids in Liquid Biopsies

International Journal of Molecular Sciences ◽

10.3390/ijms20030704 ◽

2019 ◽

Vol 20 (3) ◽

pp. 704 ◽

Cited By ~ 14

Author(s):

Aoife Ward Gahlawat ◽

Judith Lenhardt ◽

Tania Witte ◽

Denise Keitel ◽

Anna Kaufhold ◽

...

Keyword(s):

Nucleic Acids ◽

Real Life ◽

Storage Conditions ◽

Clinical Diagnostics ◽

Blood Collection ◽

Circulating Mirnas ◽

Liquid Biopsies ◽

Long Term Storage ◽

Circulating Nucleic Acids

In the last decade, circulating nucleic acids such as microRNAs (miRNAs) and cell-free DNA (cfDNA) have become increasingly important in serving as potential novel biomarkers for a variety of human diseases. If cell-free nucleic acids are to become routinely used in diagnostics, the difference in plasma miRNA and cfDNA levels between healthy and diseased subjects must exceed pre-analytical and analytical variability. Until now, few studies have addressed the time limitations of pre-processing or explored the potential use of long-term blood storage tubes, which might need to be implemented in real-life diagnostics. In this study, we analyzed the stability of four breast cancer-associated miRNAs and two cancer-associated genes under various storage conditions, to test their limitations for potential application in clinical diagnostics. In two consecutive experiments, we tested the limits of conventional EDTA tubes, as well as long-term storage blood collection tubes (BCTs) from four different manufacturers. We found that circulating miRNAs are relatively stable when stored in EDTA monovettes for up to 12 h before processing. When stored in BCTs, circulating miRNAs and cfDNA are stable for up to 7 days, depending on the manufacturer. Norgen tubes were superior for cfDNA yield, while Streck tubes performed the worst in our study with hemolysis induction. In conclusion, plasma prepared from whole blood is suitable for the quantification of both cf-miRNAs and cfDNA simultaneously.

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Preparation And elemental analysis of ancient fibers

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100126846 ◽

1987 ◽

Vol 45 ◽

pp. 410-411

Author(s):

Allen Angel ◽

Kathryn A. Jakes

Keyword(s):

Cross Sections ◽

Physical Structure ◽

Energy Dispersive ◽

Archaeological Sites ◽

X Ray ◽

Long Term Storage ◽

Backscattered Electron ◽

Electron Imaging

Fabrics recovered from archaeological sites often are so badly degraded that fiber identification based on physical morphology is difficult. Although diagenetic changes may be viewed as destructive to factors necessary for the discernment of fiber information, changes occurring during any stage of a fiber's lifetime leave a record within the fiber's chemical and physical structure. These alterations may offer valuable clues to understanding the conditions of the fiber's growth, fiber preparation and fabric processing technology and conditions of burial or long term storage (1).Energy dispersive spectrometry has been reported to be suitable for determination of mordant treatment on historic fibers (2,3) and has been used to characterize metal wrapping of combination yarns (4,5). In this study, a technique is developed which provides fractured cross sections of fibers for x-ray analysis and elemental mapping. In addition, backscattered electron imaging (BSI) and energy dispersive x-ray microanalysis (EDS) are utilized to correlate elements to their distribution in fibers.

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Modelling of Moisture Movement in Wood during Outdoor Storage

Nonlinear Analysis Modelling and Control ◽

10.15388/na.2001.6.1.15210 ◽

2001 ◽

Vol 6 (2) ◽

pp. 3-14 ◽

Cited By ~ 23

Author(s):

R. Baronas ◽

F. Ivanauskas ◽

I. Juodeikienė ◽

A. Kajalavičius

Keyword(s):

Experimental Data ◽

Finite Difference ◽

Climatic Conditions ◽

Two Dimensional ◽

Moisture Movement ◽

Finite Difference Technique ◽

Long Term Storage ◽

Space Formulation ◽

Term Storage

A model of moisture movement in wood is presented in this paper in a two-dimensional-in-space formulation. The finite-difference technique has been used in order to obtain the solution of the problem. The model was applied to predict the moisture content in sawn boards from pine during long term storage under outdoor climatic conditions. The satisfactory agreement between the numerical solution and experimental data was obtained.

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