scholarly journals Towards high–throughput analyses of fecal samples from wildlife

2020 ◽  
pp. 171-183
Author(s):  
C. Sarabia ◽  
I. Salado ◽  
A. Cornellas ◽  
A. Fernández-Gil ◽  
C. Vilà ◽  
...  
Keyword(s):  
Dna Extraction ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Low Cost ◽  
Molecular Ecology ◽  
Extraction Methods ◽  
Success Rates ◽  
Sample Collection ◽  
Field Sampling ◽  
Fecal Samples

High–throughput sequencing offers new possibilities in molecular ecology and conservation studies. However, its potential has not yet become fully exploited for noninvasive studies of free–ranging animals, such as those based on feces. High–throughput sequencing allows sequencing of short DNA fragments and could allow simultaneous genotyping of a very large number of samples and markers at a low cost. The application of high throughput genotyping to fecal samples from wildlife has been hindered by several labor–intensive steps. We evaluate alternative protocols which could allow higher throughput for two of these steps: sample collection and DNA extraction. Two different field sampling and seven different DNA extraction methods are tested here on grey wolf (Canis lupus) feces. There was high variation in genotyping success rates. The field sampling method based on surface swabbing performed much worse than the extraction from a fecal fragment. In addition, there is a lot of room for improvement in the DNA extraction step. Optimization of protocols can lead to very much more efficient, cheaper and higher throughput noninvasive monitoring. Selection of appropriate markers is still of paramount importance to increase genotyping success.

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

Comparing the performance of three ancient DNA extraction methods for high-throughput sequencing

2015 ◽  
Vol 16 (2) ◽  
pp. 459-469 ◽  
Author(s):  
Cristina Gamba ◽  
Kristian Hanghøj ◽  
Charleen Gaunitz ◽  
Ahmed H. Alfarhan ◽  
Saleh A. Alquraishi ◽  
...  
Keyword(s):  
Dna Extraction ◽  
High Throughput ◽  
Ancient Dna ◽  
High Throughput Sequencing ◽  
Extraction Methods ◽  
Dna Extraction Methods

scholarly journals Monitoring of four DNA extraction methods upstream of high-throughput sequencing of Anisakidae nematodes

2014 ◽  
Vol 102 ◽  
pp. 69-72 ◽  
Author(s):  
Y. Seesao ◽  
C. Audebert ◽  
V. Verrez-Bagnis ◽  
S. Merlin ◽  
M. Jérôme ◽  
...  
Keyword(s):  
Dna Extraction ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Extraction Methods ◽  
Dna Extraction Methods

DETECTION OF ALFACORONAVIRUSES, BETACORONAVIRUSES AND ASTROVIRUSES IN BAT FECAL SAMPLES FROM MOSCOW REGION

2020 ◽  
Author(s):  
E.V. Korneenko ◽  
◽  
А.E. Samoilov ◽  
I.V. Artyushin ◽  
M.V. Safonova ◽  
...  
Keyword(s):  
High Throughput ◽  
Viral Genome ◽  
High Throughput Sequencing ◽  
Pcr Amplification ◽  
Moscow Region ◽  
Fecal Samples ◽  
Genus Level ◽  
Zoonotic Viruses ◽  
Reservoir Hosts ◽  
Blastn Search

In our study we analyzed viral RNA in bat fecal samples from Moscow region (Zvenigorod district) collected in 2015. To detect various virus families and genera in bat fecal samples we used PCR amplification of viral genome fragments, followed by high-throughput sequencing. Blastn search of unassembled reads revealed the presence of viruses from families Astroviridae, Coronaviridae and Herpesviridae. Assembly using SPAdes 3.14 yields contigs of length 460–530 b.p. which correspond to genome fragments of Coronaviridae and Astroviridae. The taxonomy of coronaviruses has been determined to the genus level. We also showed that one bat can be a reservoir of several virus genuses. Thus, the bats in the Moscow region were confirmed as reservoir hosts for potentially zoonotic viruses.

Efficient High-throughput Techniques for the Analysis of Disease-Resistant Plant Varieties and Detection of Food Adulteration

2021 ◽  
Vol 23 ◽  
Author(s):  
Romesh Kumar Salgotra ◽  
Rafiq Ahmad Bhat ◽  
Deyue Yu ◽  
Javaid Akhter Bhat
Keyword(s):  
Multiplex Pcr ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Low Cost ◽  
Crop Improvement ◽  
Small Sample ◽  
Food Crops ◽  
Genomic Resources ◽  
Breeding Programmes ◽  
Next Generation Sequencing Ngs

Abstract: Over the past two decades, the advances in the next generation sequencing (NGS) platforms have led to the identification of numerous genes/QTLs at high-resolution for their potential use in crop improvement. The genomic resources generated through these high-throughput sequencing techniques have been efficiently used in screening of particular gene of interest particularly for numerous types of plant stresses and quality traits. Subsequently, the identified-markers linked to a particular trait have been used in marker-assisted backcross breeding (MABB) activities. Besides, these markers are also being used to catalogue the food crops for detection of adulteration to improve the quality of food. With the advancement of technologies, the genomic resources are originating with new markers; however, to use these markers efficiently in crop breeding, high-throughput techniques (HTT) such as multiplex PCR and capillary electrophoresis (CE) can be exploited. Robustness, ease of operation, good reproducibility and low cost are the main advantages of multiplex PCR and CE. The CE is capable of separating and characterizing proteins with simplicity, speed and small sample requirements. Keeping in view the availability of vast data generated through NGS techniques and development of numerous markers, there is a need to use these resources efficiently in crop improvement programmes. In summary, this review describes the use of molecular markers in the screening of resistance genes in breeding programmes and detection of adulterations in food crops using high-throughput techniques.

scholarly journals Current Perspectives on High-Throughput Sequencing (HTS) for Adventitious Virus Detection: Upstream Sample Processing and Library Preparation

Viruses ◽  
2018 ◽  
Vol 10 (10) ◽  
pp. 566 ◽  
Author(s):  
Siemon Ng ◽  
Cassandra Braxton ◽  
Marc Eloit ◽  
Szi Feng ◽  
Romain Fragnoud ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Nucleic Acids ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Virus Detection ◽  
Extraction Methods ◽  
Control Measures ◽  
Acid Extraction ◽  
Library Preparation ◽  
Sample Processing

A key step for broad viral detection using high-throughput sequencing (HTS) is optimizing the sample preparation strategy for extracting viral-specific nucleic acids since viral genomes are diverse: They can be single-stranded or double-stranded RNA or DNA, and can vary from a few thousand bases to over millions of bases, which might introduce biases during nucleic acid extraction. In addition, viral particles can be enveloped or non-enveloped with variable resistance to pre-treatment, which may influence their susceptibility to extraction procedures. Since the identity of the potential adventitious agents is unknown prior to their detection, efficient sample preparation should be unbiased toward all different viral types in order to maximize the probability of detecting any potential adventitious viruses using HTS. Furthermore, the quality assessment of each step for sample processing is also a critical but challenging aspect. This paper presents our current perspectives for optimizing upstream sample processing and library preparation as part of the discussion in the Advanced Virus Detection Technologies Interest group (AVDTIG). The topics include: Use of nuclease treatment to enrich for encapsidated nucleic acids, techniques for amplifying low amounts of virus nucleic acids, selection of different extraction methods, relevant controls, the use of spike recovery experiments, and quality control measures during library preparation.

Comparison of High-throughput and Manual DNA Extraction Methods for the Qualitative and Quantitative Analysis of MON810 Maize

2014 ◽  
Vol 28 (3) ◽  
pp. 232-249 ◽  
Author(s):  
Katarzyna Grelewska-Nowotko ◽  
Jarosław Nowosielski ◽  
Magdalena Żurawska-Zajfert ◽  
Paweł Częstobor Czembor ◽  
Sławomir Sowa
Keyword(s):  
Quantitative Analysis ◽  
Dna Extraction ◽  
High Throughput ◽  
Extraction Methods ◽  
Qualitative And Quantitative Analysis ◽  
Qualitative And Quantitative ◽  
Dna Extraction Methods ◽  
Mon810 Maize

scholarly journals A novel post hoc method for detecting index switching finds no evidence for increased switching on the Illumina HiSeq X

2017 ◽  
Author(s):  
Gregory L. Owens ◽  
Marco Todesco ◽  
Emily B. M. Drummond ◽  
Sam Yeaman ◽  
Loren H. Rieseberg
Keyword(s):  
Allele Frequency ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Sequence Data ◽  
Molecular Ecology ◽  
Whole Genome Shotgun ◽  
Whole Genome ◽  
Illumina Hiseq ◽  
Illumina Hiseq Platform ◽  
Post Hoc

AbstractHigh throughput sequencing using the Illumina HiSeq platform is a pervasive and critical molecular ecology resource, and has provided the data underlying many recent advances. A recent study has suggested that ‘index switching’, where reads are misattributed to the wrong sample, may be higher in new versions of the HiSeq platform. This has the potential to invalidate both published and in-progress work across the field. Here, we test for evidence of index switching in an exemplar whole genome shotgun dataset sequenced on both the Illumina HiSeq 2500, which should not have the problem, and the Illumina HiSeq X, which may. We leverage unbalanced heterozygotes, which may be produced by index switching, and ask whether the under-sequenced allele is more likely to be found in other samples in the same lane than expected based on the allele frequency. Although we validate the sensitivity of this method using simulations, we find that neither the HiSeq 2500 nor the HiSeq X have evidence of index switching. This suggests that, thankfully, index switching may not be a ubiquitous problem in HiSeq X sequence data. Lastly, we provide scripts for applying our method so that index switching can be tested for in other datasets.

scholarly journals Comparison of the efficiency of some of the most usual DNA extraction methods for woody plants in different tissues of Vitis vinifera L.

OENO One ◽  
2013 ◽  
Vol 47 (4) ◽  
pp. 227 ◽  
Author(s):  
Gemma Marsal ◽  
Núria Boronat ◽  
Joan Miquel Canals ◽  
Fernando Zamora ◽  
Francesca Fort
Keyword(s):  
Vitis Vinifera ◽  
Dna Extraction ◽  
Woody Plants ◽  
Extraction Method ◽  
Low Cost ◽  
Handling Time ◽  
Extraction Methods ◽  
Original Method ◽  
Dna Extraction Method ◽  
Functional Dna

<p style="text-align: justify;"><strong>Aim</strong>: To compare different methods for extracting DNA from non-recalcitrant and recalcitrant tissues of <em>Vitis vinifera</em> woody plants and propose a modification of a previously published method to reduce the time and cost of extraction.</p><p style="text-align: justify;"><strong>Methods and results</strong>: DNA was extracted from young and mature leaves as well as from stems and seeds using some of the most common methods of DNA isolation and two commercial kits. Another commercial kit, which does not require DNA extraction prior to PCR, was also used. Only two methods provided adequate results in all tissues. Other methods were only applicable to some tissues and some did not yield any functional DNA in any tissue. A modification of the method reported by Marsal <em>et al</em>. (2011) is proposed to reduce handling time and cost.</p><p style="text-align: justify;"><strong>Conclusion</strong>: All of the methods studied here use a surfactant to improve the extractions. For DNA extraction from recalcitrant tissues to be optimal, it is best to use a combination of dodecyltrimethylammonium bromide (DTAB) and cetyltrimethylammonium bromide (CTAB). The changes made to the protocol reported by Marsal <em>et al</em>. (2011) enable functional DNA to be obtained from leaves in only 90 minutes and at very low cost (17 €/8 samples). However, this method cannot adequately isolate DNA from recalcitrant tissues (stems and seeds) and so, for this type of sample, we would recommend using the original method.</p><p style="text-align: justify;"><strong>Significance and impact of the study</strong>: Nowadays, handling time and cost are key factors in selecting the most suitable DNA extraction method. This study compares not only the effectiveness of the various methods but also the handling time and cost. It also proposes a modification of the fastest and most economic DNA extraction method for leaves so that handling time and processing cost will be reduced even further.</p>

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