Sample Preservation Methods Research Articles

Tenfold difference in DNA recovery rate: systematic comparison of whole blood vs. dried blood spot sample collection for malaria molecular surveillance

BackgroundMolecular and genomic surveillance is becoming increasingly used to track malaria control and elimination efforts. Blood samples can be collected as whole blood and stored at − 20 °C until DNA extraction, or as dried blood spots (DBS), circumventing the need for a cold chain. Despite the wide use of either method, systematic comparisons of how the method of blood sample preservation affects the limit of detection (LOD) of molecular diagnosis and the proportion of DNA recovered for downstream applications are lacking.MethodsExtractions based on spin columns, magnetic beads, Tween-Chelex, and direct PCR without prior extraction were compared for whole blood and dried blood spots (DBS) using dilution series of Plasmodium falciparum culture samples. Extracted DNA was quantified by qPCR and droplet digital PCR (ddPCR).ResultsDNA recovery was 5- to 10-fold higher for whole blood compared to DBS, resulting in a 2- to 3-fold lower LOD for both extraction methods compared to DBS. For whole blood, a magnetic bead-based method resulted in a DNA recovery rate of 88–98% when extracting from whole blood compared to 17–33% for a spin-column based method. For extractions from DBS, the magnetic bead-based method resulted in 8–20% DNA recovery, while the spin-column based method resulted in only 2% DNA recovery. The Tween-Chelex method was superior to other methods with 15–21% DNA recovery, and even more sensitive than extractions from whole blood samples. The direct PCR method was found to have the lowest LOD overall for both, whole blood and DBS.ConclusionsPronounced differences in LOD and DNA yield need to be considered when comparing prevalence estimates based on molecular methods and when selecting sampling protocols for other molecular surveillance applications.

Fish eDNA detection and its technical optimization: A case study of Acanthopagrus latus

The development of a standardized eDNA detection process is the primary step in improving the accuracy and efficiency of eDNA detection. In this study, primers and probes with high specificity were selected to identify the COI gene of Acanthopagrus latus. Through experiments on the influence of different water quantities, methods of water sample preservation and water bathing times on the result of eDNA detection, the accuracy of this method for extracted water samples was improved. Specifically, a water bathing time of 6 h provided an optimal eDNA concentration from the water sample. After 6 h, the concentration began to decrease, so 6 h was determined to be the best water bathing time for A. latus. Five water extraction volumes (250 mL, 500 mL, 1 L, 2 L, and 3 L) were tested, and there was a positive correlation between water extraction volume and the DNA concentration in the water sample. Different water sample preservation methods were also compared, and it was found that at ≤7 d, the concentration obtained with the cryopreservation method for different water extraction volumes was higher than that obtained with the ethanol preservation method. In this study, we established and optimized a technical procedure for eDNA-based detection of A. latus in aquatic environments. We hope to apply this method in field investigations and provide a reference for the study of eDNA in other fishes.

Effects of Stool Sample Preservation Methods on Gut Microbiota Biodiversity: New Original Data and Systematic Review with Meta-Analysis

Effects of Stool Sample Preservation Methods on Gut Microbiota Biodiversity: New Original Data and Systematic Review with Meta-Analysis

Simple solution to preserve plant samples for microbiome analyses.

Culture-independent survey techniques are fundamental tools when assessing plant microbiomes. These methods rely on DNA that is carefully preserved after collecting samples to achieve meaningful results. Immediately freezing samples to -80°C after collection is considered one of the most robust methods for preserving samples before DNA extraction but is often impractical. Preservation solutions can solve this problem, but commercially available products are expensive, and there is limited data comparing their efficacy with other preservation methods. In this study, we compared the impact of three methods of sample preservation on plant microbiome surveys: (1) RNAlater, a proprietary preservative, (2) a home-made salt-saturated dimethyl sulphoxide preservation solution (DESS), and (3) freezing at -80°C. DESS-preserved samples, stored at room temperature for up to four weeks, did not show any significant differences to samples frozen at -80°C, while RNAlater inflated bacterial alpha diversity. Preservation treatments did not distinctively influence fungal alpha diversity. Our results demonstrate that DESS is a versatile and inexpensive preservative of DNA in plant material for diversity analyses of fungi and bacteria.

A Universal Approach to Molecular Identification of Rumen Fluke Species Across Hosts, Continents, and Sample Types.

Rumen fluke are parasitic trematodes that affect domestic and wild ruminants across a wide range of countries and habitats. There are 6 major genera of rumen fluke and over 70 recognized species. Accurate species identification is important to investigate the epidemiology, pathophysiology and economic impact of rumen fluke species but paramphistomes are morphologically plastic, which has resulted in numerous instances of misclassification. Here, we present a universal approach to molecular identification of rumen fluke species, including different life-cycle stages (eggs, juvenile and mature fluke) and sample preservation methods (fresh, ethanol- or formalin-fixed, and paraffin wax-embedded). Among 387 specimens from 173 animals belonging to 10 host species and originating from 14 countries on 5 continents, 10 rumen fluke species were identified based on ITS-2 intergenic spacer sequencing, including members of the genera Calicophoron, Cotylophoron, Fischeroedius, Gastrothylax, Orthocoelium, and Paramphistomum. Pairwise comparison of ITS-2 sequences from this study and GenBank showed >98.5% homology for 80% of intra-species comparisons and <98.5% homology for 97% of inter-species comparisons, suggesting that some sequence data may have been entered into public repositories with incorrect species attribution based on morphological analysis. We propose that ITS-2 sequencing could be used as a universal tool for rumen fluke identification across host and parasite species from diverse technical and geographical origins and form the basis of an international reference database for accurate species identification.

Investigating the association between the urinary microbiome and bladder cancer: An exploratory study

IntroductionWe sought to investigate the association between the urinary microbiome and bladder cancer, including the difference between nonmuscle-invasive (NMIBC) and muscle-invasive (MIBC) bladder cancer, and Bacillus Calmette Guerin (BCG) responsive vs. BCG-refractory NMIBC. MethodsUrine specimens were collected from consecutive patients with bladder cancer and healthy volunteers. Urine samples were analyzed using 16S rRNA sequencing to identify and compare any present bacteria. Alteration in the urinary microbiome was described in terms of alpha (diversity of populations within a sample) and beta diversities (differences between populations among different samples). Analyses were corrected for age, sex, method of sample preservation, and method of collection (mid-stream catch vs. catheterized urine). ResultsFifty-three samples (43 patients with bladder cancer, and 10 controls) were included. For bladder cancer patients, mean age was 70 years, 7 (16%) were females; and 29 (67%) had NMIBC. Among patients with NMIBC, 11 (38%) patients received BCG, 6 of which had recurrence or progression after a median follow up of 13 months. Comparing the microbiome of bladder cancer patients vs. healthy controls, beta-diversity was significantly different, with Actinomyces, Achromobacter, Brevibacterium, and Brucella significantly more abundant in urine samples of bladder cancer patients. Comparing NMIBC and MIBC, Hemophilus and Veillonella were significantly more abundant in urine of MIBC patients, while Cupriavidus was significantly more abundant in NMIBC patients. Among NMIBC patients, Serratia and Brochothrix, Negativicoccus, Escherichia-Shigella, and Pseudomonas were significantly more abundant in patients who responded to BCG in comparison to those who did not. ConclusionUrinary microbiome varied between patients with bladder cancer and healthy controls. Moreover, urinary microbial profiles differed among patient with NMIBC vs. MIBC, and among BCG responsive vs. BCG refractory NMIBC.

Prokaryotic and Fungal Characterization of the Facilities Used to Assemble, Test, and Launch the OSIRIS-REx Spacecraft.

To characterize the ATLO (Assembly, Test, and Launch Operations) environment of the OSIRIS-REx spacecraft, we analyzed 17 aluminum witness foils and two blanks for bacterial, archaeal, fungal, and arthropod DNA. Under NASA’s Planetary Protection guidelines, OSIRIS-REx is a Category II outbound, Category V unrestricted sample return mission. As a result, it has no bioburden restrictions. However, the mission does have strict organic contamination requirements to achieve its primary objective of returning pristine carbonaceous asteroid regolith to Earth. Its target, near-Earth asteroid (101955) Bennu, is likely to contain organic compounds that are biologically available. Therefore, it is useful to understand what organisms were present during ATLO as part of the larger contamination knowledge effort—even though it is unlikely that any of the organisms will survive the multi-year deep space journey. Even though these samples of opportunity were not collected or preserved for DNA analysis, we successfully amplified bacterial and archaeal DNA (16S rRNA gene) from 16 of the 17 witness foils containing as few as 7 ± 3 cells per sample. Fungal DNA (ITS1) was detected in 12 of the 17 witness foils. Despite observing arthropods in some of the ATLO facilities, arthropod DNA (COI gene) was not detected. We observed 1,009 bacterial and archaeal sOTUs (sub-operational taxonomic units, 100% unique) and 167 fungal sOTUs across all of our samples (25–84 sOTUs per sample). The most abundant bacterial sOTU belonged to the genus Bacillus. This sOTU was present in blanks and may represent contamination during sample handling or storage. The sample collected from inside the fairing just prior to launch contained several unique bacterial and fungal sOTUs that describe previously uncharacterized potential for contamination during the final phase of ATLO. Additionally, fungal richness (number of sOTUs) negatively correlates with the number of carbon-bearing particles detected on samples. The total number of fungal sequences positively correlates with total amino acid concentration. These results demonstrate that it is possible to use samples of opportunity to characterize the microbiology of low-biomass environments while also revealing the limitations imposed by sample collection and preservation methods not specifically designed with biology in mind.

Effects of soil preservation for biodiversity monitoring using environmental DNA.

Environmental DNA (eDNA) metabarcoding is becoming a key tool for biodiversity monitoring over large geographical or taxonomic scales and for elusive taxa such as soil organisms. Increasing sample sizes and interest in remote or extreme areas often require the preservation of soil samples and thus deviations from optimal standardized protocols. However, we still ignore the impact of different methods of soil sample preservation on the results of metabarcoding studies and there is no guideline for best practices so far. Here, we assessed the impact of four methods of soil sample preservation that can be conveniently used also in metabarcoding studies targeting remote or difficult to access areas. Tested methods include: preservation at room temperature for 6hr, preservation at 4°C for 3days, desiccation immediately after sampling and preservation for 21days, and desiccation after 6hr at room temperature and preservation for 21days. For each preservation method, we benchmarked resulting estimates of taxon diversity and community composition of three different taxonomic groups (bacteria, fungi and eukaryotes) in three different habitats (forest, river bank and grassland) against results obtained under ideal conditions (i.e., extraction of eDNA immediately after sampling). Overall, the different preservation methods only marginally impaired results and only under certain conditions. When rare taxa were considered, we detected small but significant changes in molecular operational taxonomic units (MOTU) richness of bacteria, fungi and eukaryotes across treatments, but MOTU richness was similar across preservation methods if rare taxa were not considered. All the approaches were able to identify differences in community structure among habitats, and the communities retrieved using the different preservation conditions were extremely similar. We propose guidelines on the selection of the optimal soil sample preservation conditions for metabarcoding studies, depending on the practical constraints, costs and ultimate research goals.

Storage Procedures Affect pH, Electrical Conductivity, and Nutrient Concentrations of Pour-through Leachate from Pine Bark and Peat-based Substrates

The pour-through (PT) method is used in greenhouse and nursery production to monitor nutrient availability in soilless substrates. Efficacy of this method is based on the assumption that chemical properties of extracted solutions remain stable from the moment of collection until analysis. Extracted substrate solution can be analyzed directly in the greenhouse or sent to laboratories for complete nutritional analysis; thus, proper sample preservation methods (e.g., filtration and low temperatures) are critical for reducing sample contamination or degradation during storage. However, evidence of how these preservation methods affect chemical characteristics of PT samples is limited. The objective of this study was to evaluate the effect of storage time, storage temperature, and filtration of PT samples on pH, electrical conductivity (EC), and nutrient concentrations from pine bark– and peat-based substrates. PT extracts were obtained from liquid-fertilized fallow pots of either 100% milled pine bark (Expt. 1) or a 4 sphagnum peat: 1 perlite (by volume) substrate (Expt. 2). Aliquots of PT extract were either filtered or nonfiltered and then stored in plastic bottles at −22, 4, or 20 °C. EC, pH, and nutrient concentrations were analyzed at 0, 1, 7, and 30 days after PT sample collection. EC and pH in PT extracts of peat and pine bark, respectively, changed 1 day after collection. Storage time had the greatest effect on nutrient concentrations of samples stored at 20 °C. However, at day 30, nutrient concentrations had also changed in samples stored at 4 and −22 °C. Analytes that fluctuated most in both experiments and across all preservation treatments were dissolved organic carbon, total dissolved nitrogen, NO3−-N, and PO43−-P, whereas Ca2+, Mg2+, and SO42−-S were more stable in PT samples. This research suggests EC and pH should be analyzed immediately, whereas samples requiring nutrient analysis should be filtered immediately after collection, stored at 4 or −22 °C (preferably −22 °C), and analyzed within 7 days of collection.

Sample Preservation and Plant Sex Prediction in White Guinea yam (Dioscorea rotundata Poir.)

Introduction: Yam (Dioscorea spp.) is an economically important staple food in tropical regions, especially for people in West Africa. Understanding of the flowering behavior of the crop to determine potential manipulation available to accomplish crop improvement at early stage remain key challenge in the yam breeding. The methods that reliably yield quality DNA and distinguishing sex type at the early stage of growth have been a challenge in yam genetics and breeding studies. This study assessed the effect of sample preservation methods on DNA quantity and quality during extraction and potential of DNA marker to diagnose plant sex at the early seedling stage in white Guinea yam. Materials and Methods: Five sample preservation methods were assessed for quality DNA extraction during field leaf tissue collection, namely liquid nitrogen, dry ice, silica gel, 95% ethanol, and oven drying. The predicted sex at the seedling stage using the molecular marker was further validated with the visual score for the sex phenotype at the flowering stage. Results: According to the findings of the present study, the DNA extracted from leaf samples preserved in liquid nitrogen, silica gel, dry ice, and oven drying methods were higher in molecular weights than samples stored in ethanol solution. Yam plant sex diagnosis with the DNA marker (sp16) identified a higher proportion of ZW genotypes (female or monoecious phenotypes) than the ZZ genotypes (male phenotypes) in the studied materials with 74% prediction accuracy. Conclusions: The results from this study provided valuable insights on suitable sample preservation methods for quality DNA extraction and the potential of DNA marker sp16 to predict sex in white Guinea yam.

Effects of different sample preservation methods on the lymphocyte subset detection by flow cytometry

Objective To discuss the effects of sample storage time and temperature on lymphocyte subsets detected by flow cytometry. Methods Use flow cytometry to detect lymphocyte subsets of a total of 53 blood samples from hospitalized and out-patient patients in Qinghai Provincial People′s Hospital from October 26, 2018 to March 30, 2019. The test was completed within 4 hours after sample collection, which is the control group. The treatment groups are as follows: pretreatment was completed within 4 hours and detected after saving samples at room temperature (group A) for 24 and 36 hours; the tests were performed after keeping samples at room temperature (group B) for 24, 48, 72 hours; completed detection after preserving samples in 4 ℃ condition (group C) for 24, 48, 72 hours. Results In treatment group A, there was no significant difference in the results of lymphocyte subsets detected after 24 hours of storage compared with the control group(P>0.05); after 36 h of storage, CD3+and CD3+CD8+T lymphocytes percentages were significantly increased [(74.28±11.31)% vs (73.78±11.33)%, (32.15±14.82)% vs (31.00±14.79)%; all P 0.05); after 48 hours of storage, CD19+cells percentage were observably reduced [(15.60±12.09)% vs (16.11±12.38)%; P 0.05); after 72 hours of storage, CD3+and CD3+CD8+T cells percentages elevated significantly [(75.78±11.18)% vs (73.78±11.33)%, (32.57±14.90)% vs (31.00±14.79)%; all P 0.05); after 48 hours of storage, CD3+CD8+T cells percentage elevated obviously [(32.03±14.95)% vs (31.00±14.79)%; P<0.05] and CD19+cells percentage decreased markedly [(15.32±11.97)% vs (16.11±12.38)%; P<0.05]; after 72 hours of storage, CD3+and CD3+CD8+T cells percentages were significantly increased [(75.63±11.08)% vs (73.78±11.33)%, (32.62±14.98)% vs (31.00±14.79)%; all P<0.05], while CD56+and CD19+cells percentages were reduced observably [(8.21±6.52)% vs (9.02±6.80)%, (14.83±11.79)% vs (16.11±12.38)%; all P<0.05]. Conclusions The blood samples can be kept at room temperature and the testshould be completed within 48 h if the detection of lymphocyte subsets by flow cytometry cannot be performed in time; for the samples that have been pretreated, detection can be completed after saving at room temperature for 24 h, and the above treatments had no significant effect on the inspection results. Key words: Lymphocyte subsets; Flow cytometry; Sample preservation

Could biological tissue preservation methods change chemical elements proportion measured by energy dispersive X-ray spectroscopy?

Energy dispersive X-ray spectroscopy (EDS) is a powerful technical tool used in the biomedical field to investigate the proportion of chemical elements of interest in research, such as heavy metal bioaccumulation and the enzymatic cofactors and nanoparticle therapy in various pathologies. However, the correct evaluation of the proportion of the elements is subject to some factors, including the method of sample preservation. In this study, we seek to investigate the effect of biological tissue preservation methods on the proportion of chemical elements obtained by the EDS methodology. For such, we used EDS to measure the proportion of chemical elements with biomedical interest in preserved livers, using three common methods for preserving biological tissues: (a) freezing, (b) paraformaldehyde fixative solution, and (c) Karnovsky solution. We found an increased level of sodium and reduced contents of potassium and copper in samples fixed in fixative solutions, when compared to frozen samples (p< 0.05). Our data indicate that preservation methods can change the proportion of chemical elements in biological samples, when measured by EDS. Frozen preservation should be preferred to retain the actual chemical content of samples and allow a correct assessment of the proportion of their elements.

Methodologies for Plasmopara halstedii Research

Abstract The objective of this work was to find practical procedures to overcome methodological drawbacks encountered during studies on sunflower downy mildew. Techniques for recovering living isolates of Plasmopara halstedii from the field and for the preservation of infected leaf samples for further molecular analysis were developed. A Polymerase Chain Reaction (PCR)-based test for the detection of P. halstedii in sunflower leaves and a method to remove azoxystrobin from fungicide-treated seeds are proposed. In situ-inoculations of pre-germinated seeds allowed the recovery of living isolates from the field. Three sample-preservation methods were evaluated (silica, heating and lyophilization) resulting in high yield and quality of the DNA extract. It was detected the presence of the pathogen in symptomless leaves through PCR using molecular markers based on expressed sequence tags. A treatment using sodium hypochlorite is recommended for the removal of azoxystrobin from fungicide treated seeds.

Method for DNA Isolation From Sweetpotato Weevil (Coleoptera: Curculionidae) Collected in Pheromone-Baited Traps.

This study provides a protocol for the isolation of high-quality DNA from sweetpotato weevils (Cylas formicarius elegantulus (Summers)) collected from pheromone-baited aerial funnel traps. This study was based on our discovery that a 2-wk collection interval of sweetpotato weevils from pheromone traps did not permit isolation of intact high-quality genomic DNA. To test the effect of collection methods, i.e., sample collection interval and preservation method, on quality of isolated DNA, we placed freshly killed male sweetpotato weevils into aerial funnel traps in the field and removed subsamples at several times thereafter. DNA yield from freshly isolated (day = 0) samples was significantly greater than samples preserved in 70% ethanol or at -20°C, whereas there was no difference between 70% ethanol and -20°C storage. Likewise, DNA yield from freshly isolated (day = 0) samples was significantly greater than for later sampling times. Quality assessment of genomic DNA through gel electrophoresis and polymerase chain reaction (PCR) indicated isolation of high molecular weight DNA for all samples collected at t ≤ 7 d, but that DNA quality was degraded by 14 d. Our goal was to develop a reliable method for isolation of genomic DNA from field-collected sweetpotato weevil suitable for direct use in PCR. We discovered that it is critical to collect specimens from traps at an interval of 1 wk or less. Our findings allow for scheduling of sampling at reasonable intervals without the need for special materials. This has the added benefit of allowing individuals without special training to collect and prepare sweetpotato weevil specimens for genetic studies.

Seafood sleuthing: How citizen science contributed to the largest market study of seafood mislabeling in the U.S. and informed policy

A seafood fraud campaign was launched by an ocean conservation group to increase transparency in global seafood supply chains by enacting policies on full chain boat-to-plate traceability for all seafood sold in the U.S. As part of this campaign, online members of the group were recruited to document and collect commercial seafood samples as part of a large investigation of U.S. seafood mislabeling, specifically species substitution. Following an iterative project design including several rounds of pilot testing of sample preservation methods and outreach materials, 1058 of the more than 55,000 members solicited signed up to be a “seafood sleuth” and were mailed seafood testing kits, containing supplies to submit two fish samples of their choice. On average, 33.4% (353/1058) of these citizen scientists in 11 metropolitan areas returned kits that contained 631 samples, or nearly half of the 1263 samples collected in the overall study. Assessment of the quality of citizen science data revealed comparable rates of sample integrity, data completeness and mislabeling compared to samples and data collected by trained scientists. Citizen science outreach provided a more informed and engaged online member population, who continued to take actions to advance seafood traceability policies with their decision makers. Citizen science outreach was an integral part of a successful campaign that included science, communication strategies to garner mass media attention and advocacy to promote seafood traceability which resulted in the first seafood traceability regulation in the U.S.

High purity high yield tandem B and T helper cell isolation for qRT-PCR analysis suitable for basically equipped laboratories

BackgroundMalaria is still a major health problem in sub-Saharan Africa and south-east Asia, but research on malaria in low-income countries can be a challenge due to the lack of laboratory equipment. In addition, severe malaria mainly affects very young children, which limits the amount of blood available for research purposes. Thus, there is a need for protocols that yield a maximum of information from a minimum amount of blood, which are operable in basically equipped laboratories.ResultsA protocol for tandem B and T helper (Th) cell isolation directly from whole blood, and a freezer-independent sample preservation method compatible with the warm and humid climate of malaria regions was established and validated. The protocol thereby circumvents the need of high-technology centrifuges and unimpeachable power supply for peripheral blood mononuclear cell isolation. Both purity and yield are excellent. Depending on the expression level of the genes of interest, between 2 and 5 ml of blood are adequate for reliable qRT-PCR results from both B and Th cells of healthy paediatric donors as well as paediatric malaria patients.ConclusionThis protocol for high purity high yield B cell and Th cell isolation and sample storage for subsequent qRT-PCR analysis from a minimal amount of blood is contrivable with basic equipment and independent of continuous power supply. Thus, it is likely to be of avail for many scientists performing malaria research in rural institutes or hospitals, and thus in countries where malaria is most prevalent.

The impact of cell fixation on coherent anti-stokes Raman scattering signal intensity in neuronal and glial cell lines.

A number of studies require sample fixation, aimed to preserve cells in a physiological state with minimal changes of morphology and intracellular molecular content. Sample fixation may significantly distort experimental data, which makes the data interpretation process more challenging. It is particularly important for study of lipid-related diseases, where the biochemical and morphological characteristics of the cells need to be well preserved for an accurate data analysis. This study investigates the effects of formaldehyde and ethanol (EtOH) fixatives on coherent anti-stokes Raman scattering (CARS) signal of proteins and lipids in major cellular compartments of neuronal and glial cells. We found that both fixatives induce alteration of proteins and lipids signal in studied cell lines. Furthermore, the impact of sample preservation methods on CARS signal varies between cell lines. For instance, our data reveals that EtOH fixation induces ~45% increase of CARS signal of proteins in the nucleolus of neuronal cells and ~35% decrease of CARS signal in glial cells. The results indicate that aldehyde fixation is a preferable method for preservation of neuronal and glial cells prior to CARS imaging, as it less affects both CARS signal and intracellular distribution of proteins and lipids.

A novel affordable reagent for room temperature storage and transport of fecal samples for metagenomic analyses

BackgroundThe number of large-scale studies on the gut microbiota in human cohorts is rapidly increasing. However, the few and expensive options for storage of fecal samples at room temperature have been an obstacle for large-scale metagenomic studies and the development of clinical/commercial personal metagenomic sequencing.ResultsIn this study, we systematically tested a novel N-octylpyridinium bromide-based fecal sample preservation method and compared it with other currently used storage methods. We found that the N-octylpyridinium bromide-based method enabled preservation of the bacterial composition in fecal samples transported and stored at room temperature for up to at least 14 days.ConclusionsWe describe a novel chemical stabilizer that allows cost-effective transportation and storage at room temperature for several days with preservation of bacterial composition. This method will facilitate sample collection even in remote area and also enable transport via normal commercial transportation routes.

Optimization of disinfection by-product analysis methods for IMO G9 approval

This study assessed the methods for analyzing disinfection by-products (DBPs) to determine which were most suitable for ballast water in an approval test according to the Procedure for Approval of Ballast Water Management Systems that make use of Active Substances (G9). The existing analysis methods are optimized for drinking water. Therefore, it is necessary to assess the characteristics of ballast water, which has high levels of total residual oxidants (TROs) and salinity, prior to applying the existing methods. Sample preservation, pre-treatment and instrumental analysis methods were summarized based on certified test methods and the G9 final approval reports. Following the assessment, applicable methods were selected in consideration of the matrix effect arising from the high levels of TROs and salinity. The applicability was assessed using seawater and brackish water. The results are expected to be applied to the G9 test as well as in investigations of DBPs in ballast water.

Sample Preservation, DNA or RNA Extraction and Data Analysis for High-Throughput Phytoplankton Community Sequencing.

Phytoplankton is the basis for aquatic food webs and mirrors the water quality. Conventionally, phytoplankton analysis has been done using time consuming and partly subjective microscopic observations, but next generation sequencing (NGS) technologies provide promising potential for rapid automated examination of environmental samples. Because many phytoplankton species have tough cell walls, methods for cell lysis and DNA or RNA isolation need to be efficient to allow unbiased nucleic acid retrieval. Here, we analyzed how two phytoplankton preservation methods, three commercial DNA extraction kits and their improvements, three RNA extraction methods, and two data analysis procedures affected the results of the NGS analysis. A mock community was pooled from phytoplankton species with variation in nucleus size and cell wall hardness. Although the study showed potential for studying Lugol-preserved sample collections, it demonstrated critical challenges in the DNA-based phytoplankton analysis in overall. The 18S rRNA gene sequencing output was highly affected by the variation in the rRNA gene copy numbers per cell, while sample preservation and nucleic acid extraction methods formed another source of variation. At the top, sequence-specific variation in the data quality introduced unexpected bioinformatics bias when the sliding-window method was used for the quality trimming of the Ion Torrent data. While DNA-based analyses did not correlate with biomasses or cell numbers of the mock community, rRNA-based analyses were less affected by different RNA extraction procedures and had better match with the biomasses, dry weight and carbon contents, and are therefore recommended for quantitative phytoplankton analyses.