Modified Methods for Loading of High-Throughput DNA Extraction Plates Reduce Potential for Contamination

10.3791/61405 ◽  
2020 ◽  
Author(s):  
Gordon F. Custer ◽  
Reilly R. Dibner
Keyword(s):  
Dna Extraction ◽  
High Throughput

scholarly journals The Fellowship of the Ring Test: DNAqua-Net WG2 initiative to compare diatom metabarcoding protocols used in routine freshwater biomonitoring for standardisation

2021 ◽  
Vol 4 ◽  
Author(s):  
Valentin Vasselon ◽  
Éva Ács ◽  
Salomé Almeida ◽  
Karl Andree ◽  
Laure Apothéloz-Perret-Gentil ◽  
...  
Keyword(s):  
Dna Extraction ◽  
High Throughput ◽  
Aquatic Ecosystems ◽  
Quality Index ◽  
Ecological Status ◽  
Pcr Amplification ◽  
Ring Test ◽  
Dna Metabarcoding ◽  
Ecological Status Assessment ◽  
Status Assessment

During the past decade genetic approaches have been developed to monitor biodiversity in aquatic ecosystems. These enable access to taxonomic and genetic information from biological communities using DNA from environmental samples (e.g. water, biofilm, soil) and methods based on high-throughput sequencing technologies, such as DNA metabarcoding. Within the context of the Water Framework Directive (WFD), such approaches could be applied to assess Biological Quality Elements (BQE). These are used as indicators of the ecological status of aquatic ecosystems as part of national monitoring programs of the european network of 110,000 surface water monitoring sites with 79.5% rivers and 11% lake sites (Charles et al. 2020). A high-throughput method has the potential to increase our spatio-temporal monitoring capacity and to accelerate the transfer of information to water managers with the aim to increase protection of aquatic ecosystems. Good progress has been made with developing DNA metabarcoding approaches for benthic diatom assemblages. Technological innovation and protocol optimization have allowed robust taxonomic (species) and genetic (OTU, ESV) information to be obtained from which diatom quality indices can be calculated to infer ecological status to rivers and lakes. Diatom DNA metabarcoding has been successfully applied for biomonitoring at the scale of national river monitoring networks in several countries around the world and can now be considered technically ready for routine application (e.g. Apothéloz-Perret-Gentil et al. 2017, Bailet et al. 2019, Mortágua et al. 2019, Vasselon et al. 2019, Kelly et al. 2020, Pérez-Burillo et al. 2020, Pissaridou et al. 2021). However, protocols and methods used by each laboratory still vary between and within countries, limiting their operational transferability and the ability to compare results. Thus, routine use of DNA metabarcoding for diatom biomonitoring requires standardization of all steps of the metabarcoding procedure, from the sampling to the final ecological status assessment in order to define good practices and standards. Following previous initiatives which resulted in a CEN technical report for biofilm sampling and preservation (CEN 2018), a set of experiments was initiated during the DNAqua-Net WG2 diatom workshop (Cyprus, 2019) to focus on DNA extraction and PCR amplification steps in order to evaluate: i) the transferability and reproducibility of a protocol between different laboratories; ii) the variability introduced by different protocols currently applied by the scientific community. 19 participants from 14 countries performed DNA extraction and PCR amplification in parallel, using i) the same fixed protocol and ii) their own protocol. Experiments were performed by each participant on a set of standardized DNA and biofilm samples (river, lake, mock community). In order to specifically test the variability of DNA extraction and PCR amplification steps, all other steps of the metabarcoding process were fixed and the preparation of the Miseq sequencing was performed by only one laboratory. The variability within and between participants will be evaluated on DNA extracts quantity, taxonomic (genus, species) and genetic richness, community structure comparison and diatom quality index scores (IPS). We will also evaluate the variability introduced by different DNA extraction and PCR amplification protocols on diatom quality index scores and the final ecological status assessment. The results from this collaborative work will not serve to define “one protocol to rule them all”, but will provide valuable information to define guidelines and minimum requirements that should be considered when performing diatom metabarcoding for biomonitoring.

An easily-performed high-throughput method for plant genomic DNA extraction

2019 ◽  
Vol 569 ◽  
pp. 28-30 ◽  
Author(s):  
Deshui Yu ◽  
Ju Zhang ◽  
Guangxuan Tan ◽  
Ningshu Yu ◽  
Qiuyue Wang ◽  
...  
Keyword(s):  
Dna Extraction ◽  
High Throughput ◽  
Genomic Dna ◽  
Genomic Dna Extraction ◽  
High Throughput Method

scholarly journals Breeding synthetic varieties in annual caraway: observations on the outcrossing rate in a polycross using a high-throughput genotyping system

Euphytica ◽  
2020 ◽  
Vol 217 (1) ◽  
Author(s):  
Daniel von Maydell ◽  
Julia Brandes ◽  
Heike Lehnert ◽  
Wolfram Junghanns ◽  
Frank Marthe
Keyword(s):  
Medicinal Plant ◽  
Dna Extraction ◽  
High Throughput ◽  
Inbred Lines ◽  
High Variability ◽  
Outcrossing Rate ◽  
Breeding Method ◽  
High Throughput Method ◽  
Synthetic Varieties ◽  
Seed Yields

AbstractCaraway (Carum carvi) is an economically important spice and medicinal plant of the Apiaceae family (syn. Umbelliferrae). Farmers often favor annual cultivation of caraway. However, the annual varieties, which are currently available, do not provide satisfying seed yields. Employing heterosis can be a promising approach to increase yield. Breeding of synthetic varieties utilizes heterosis and may be the method of choice for future caraway breeding. Knowledge of the outcrossing rate is important to evaluate the effectiveness of this breeding method. However, the outcrossing rate of caraway is unknown so far. We estimated the outcrossing rate of seven inbred lines under field conditions in a neighbor-balanced polycross design. For this purpose, we implemented a high-throughput genotyping system (PACE), accompanied by a high-throughput method for DNA extraction adapted to caraway. In total, more than 1300 individual plants were genotyped. We found a high variability of lines regarding outcrossing rate and other traits associated with flowering. The outcrossing rate was on average 66.5% and ranged from 51.6 to 82%. We discussed implications of our findings on the targeted breeding method.

scholarly journals High-Throughput DNA Extraction from Old Paraffin-Embedded Biopsies

BioTechniques ◽  
1999 ◽  
Vol 27 (2) ◽  
pp. 334-338 ◽  
Author(s):  
J. Jalouli ◽  
L. Sand ◽  
B. Gustavsson ◽  
J.M. Hirsch ◽  
P.-A. Larsson
Keyword(s):  
Dna Extraction ◽  
High Throughput

High-throughput DNA extraction from forest trees

2002 ◽  
Vol 20 (4) ◽  
pp. 425-425 ◽  
Author(s):  
Mervyn Shepherd ◽  
Michael Cross ◽  
Rhonda L. Stokoe ◽  
Leon J. Scott ◽  
Megan E. Jones
Keyword(s):  
Dna Extraction ◽  
High Throughput ◽  
Forest Trees

scholarly journals Application of high-throughput sequencing (HTS) metabarcoding to diatom biomonitoring: Do DNA extraction methods matter?

2017 ◽  
Vol 36 (1) ◽  
pp. 162-177 ◽  
Author(s):  
Valentin Vasselon ◽  
Isabelle Domaizon ◽  
Frédéric Rimet ◽  
Maria Kahlert ◽  
Agnès Bouchez
Keyword(s):  
Dna Extraction ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Extraction Methods ◽  
Dna Extraction Methods
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