The development of miniplex primer sets for the analysis of degraded DNA

Species diversity of metazoan bulk samples can be rapidly assessed using cytochrome c oxidase I (COI) metabarcoding. However, in some applications often only degraded DNA is available, e.g. from poorly conserved museum specimens, environmental DNA (eDNA) filtered from water or gut content analyses. Here universal primer sets targeting only a short COI fragment are advantageous, as they often can still amplify short DNA fragments. Using PrimerMiner, we optimised two universal primer sets targeting freshwater macroinvertebrates based on NCBI and BOLD reference sequences. The fwh1 and fwh2 primer sets targeting a 178 and 205 bp region were tested in vitro by sequencing previously used freshwater macroinvertebrate mock communities as well as three monitoring samples from Romanian streams of unknown composition. They were further evaluated in silico for their suitability to amplify other insect groups. The fwh1 primer sets showed the most consistent amplification in silico and in vitro , detecting 92% of the taxa present in the mock communities, and allowing clear differentiation between the three macroinvertebrate communities from the Romanian streams. In silico analysis indicates that the short primers are likely to perform well even for non-freshwater insects. Comparing the performance of the new fwh1 primer sets to a highly degenerate primer set targeting a longer fragment (BF2+BR2) revealed that detection efficiency is slightly lower for the new primer set. Nevertheless, the shorter new primer pairs might be useful for studies that have to rely on degraded or poorly conserved DNA and thus be of importance for biomonitoring, conservation biological or molecular ecological studies. Furthermore, our study highlights the need for in silico evaluation of primer sets in order to detect design errors in primers (fwhR2) and find optimal universal primer sets for the target taxa of interest.

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Short COI markers for freshwater macroinvertebrate metabarcoding

10.7287/peerj.preprints.3037v1 ◽

2017 ◽

Author(s):

Ecaterina Edith Vamos ◽

Vasco Elbrecht ◽

Florian Leese

Keyword(s):

In Silico ◽

In Silico Analysis ◽

Degraded Dna ◽

Macroinvertebrate Communities ◽

Universal Primer ◽

Primer Sets ◽

Clear Differentiation ◽

Silico Analysis ◽

Mock Communities

Species diversity of metazoan bulk samples can be rapidly assessed using Cytochrome c oxidase I (COI) metabarcoding. However, in cases where only degraded DNA is available, e.g. from poorly conserved museum specimens, eDNA filtered from water or gut content analyses, universal primer sets that amplify only a short COI fragment are advantageous. Using PrimerMiner, we optimised two universal primer sets targeting freshwater macroinvertebrates based on NCBI and BOLD reference sequences. The fwh1 and fwh2 primer sets targeting a 178 and 205 bp region were tested in vivo by sequencing previously used freshwater macroinvertebrate mock communities of known composition and three monitoring samples from Romanian streams. They were further evaluated in silico for their suitability to amplify other insect groups. The fwh1 primer sets showed the most consistent amplification in silico and in vivo , detecting 92% of the taxa present in the mock communities, and allowing clear differentiation between the three macroinvertebrate communities from the Romanian streams. In silico analysis indicates that the short primers are likely to perform well even for non-freshwater insects. Comparing the performance of the new fwh1 primer sets to a highly degenerate primer set targeting a longer fragment (BF2/BR2) revealed that efficiency is slightly lower for the new primer set. Nevertheless, the shorter new primer pairs might be useful for studies that have to rely on degraded or poorly conserved DNA and thus be of importance for biomonitoring, conservation biological or molecular ecological studies. Furthermore, our study highlights the need for in silico evaluation of primer sets in order to detect design errors in primers (fwhR2) and find optimal universal primer sets for the target taxa of interest.

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In silico and empirical evaluation of twelve COI & 16S metabarcoding primer sets for insectivorous diet analyses

10.1101/742874 ◽

2019 ◽

Cited By ~ 2

Author(s):

Orianne Tournayre ◽

Maxime Leuchtmann ◽

Ondine Filippi-Codaccioni ◽

Marine Trillat ◽

Sylvain Piry ◽

...

Keyword(s):

In Silico ◽

Empirical Evaluation ◽

Environmental Dna ◽

Degraded Dna ◽

Taxonomic Identification ◽

Mock Community ◽

Predator Detection ◽

Primer Sets ◽

Diet Analyses

AbstractThis last decade, environmental DNA metabarcoding approaches have been developed and improved to minimize biological and technical biases; some challenges, however, remain, as the design of primers. Here we have performed a comprehensive assessment of ten COI and two 16S primer sets. We have combined in silico, in vivo-mock community of 33 arthropod taxa from 16 orders and guano analyses to identify primer sets that should maximize arthropod detection and taxonomic identification, whilst identifying bat species and minimizing labour time and cost. We have focused on two insectivorous bat species living in mixed-colonies, the greater horseshoe bat (Rhinolophus ferrumequinum) and Geoffroy’s bat (Myotis emarginatus). We have found that the level of primer degeneracy is the main factor influencing arthropod detection for in silico and mock community analyses, while the amplicon length is critical for the detection of arthropods from degraded DNA samples. Our results confirm the importance of performing predator detection and taxonomic identification, simultaneously with arthropod sequencing, as faeces samples can be contaminated by different insectivorous species. Moreover, amplifying bat DNA does not affect the primers’ capacity to detect arthropods. We therefore recommend the systematic simultaneous identification of predator and prey. Finally, we evidenced that one third of the prey occurrences are unreliable and probably not of primary interest in diet studies, which might decrease the relevance of combining several primer sets instead of using one efficient primer set. In conclusion, this study provides general criteria enabling the selection of primers whilst considering different scientific and methodological constraints.

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Validation of COI metabarcoding primers for terrestrial arthropods

PeerJ ◽

10.7717/peerj.7745 ◽

2019 ◽

Vol 7 ◽

pp. e7745 ◽

Cited By ~ 22

Author(s):

Vasco Elbrecht ◽

Thomas W.A. Braukmann ◽

Natalia V. Ivanova ◽

Sean W.J. Prosser ◽

Mehrdad Hajibabaei ◽

...

Keyword(s):

Degraded Dna ◽

Taxonomic Resolution ◽

Effect Of Temperature ◽

Gut Contents ◽

Minor Effect ◽

Malaise Trap ◽

Mock Community ◽

Terrestrial Arthropods ◽

Primer Sets ◽

Trap Sample

Metabarcoding can rapidly determine the species composition of bulk samples and thus aids biodiversity and ecosystem assessment. However, it is essential to use primer sets that minimize amplification bias among taxa to maximize species recovery. Despite this fact, the performance of primer sets employed for metabarcoding terrestrial arthropods has not been sufficiently evaluated. This study tests the performance of 36 primer sets on a mock community containing 374 insect species. Amplification success was assessed with gradient PCRs and the 21 most promising primer sets selected for metabarcoding. These 21 primer sets were also tested by metabarcoding a Malaise trap sample. We identified eight primer sets, mainly those including inosine and/or high degeneracy, that recovered more than 95% of the species in the mock community. Results from the Malaise trap sample were congruent with the mock community, but primer sets generating short amplicons produced potential false positives. Taxon recovery from both mock community and Malaise trap sample metabarcoding were used to select four primer sets for additional evaluation at different annealing temperatures (40–60 °C) using the mock community. The effect of temperature varied by primer pair but overall it only had a minor effect on taxon recovery. This study reveals the weak performance of some primer sets employed in past studies. It also demonstrates that certain primer sets can recover most taxa in a diverse species assemblage. Thus, based our experimental set up, there is no need to employ several primer sets targeting the same gene region. We identify several suitable primer sets for arthropod metabarcoding, and specifically recommend BF3 + BR2, as it is not affected by primer slippage and provides maximal taxonomic resolution. The fwhF2 + fwhR2n primer set amplifies a shorter fragment and is therefore ideal when targeting degraded DNA (e.g., from gut contents).

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Short COI markers for freshwater macroinvertebrate metabarcoding

10.7287/peerj.preprints.3037 ◽

2017 ◽

Cited By ~ 1

Author(s):

Ecaterina Edith Vamos ◽

Vasco Elbrecht ◽

Florian Leese

Keyword(s):

In Silico ◽

Detection Efficiency ◽

In Silico Analysis ◽

Environmental Dna ◽

Degraded Dna ◽

Macroinvertebrate Communities ◽

Universal Primer ◽

Primer Sets ◽

Mock Communities

Species diversity of metazoan bulk samples can be rapidly assessed using cytochrome c oxidase I (COI) metabarcoding. However, in some applications often only degraded DNA is available, e.g. from poorly conserved museum specimens, environmental DNA (eDNA) filtered from water or gut content analyses. Here universal primer sets targeting only a short COI fragment are advantageous, as they often can still amplify short DNA fragments. Using PrimerMiner, we optimised two universal primer sets targeting freshwater macroinvertebrates based on NCBI and BOLD reference sequences. The fwh1 and fwh2 primer sets targeting a 178 and 205 bp region were tested in vitro by sequencing previously used freshwater macroinvertebrate mock communities as well as three monitoring samples from Romanian streams of unknown composition. They were further evaluated in silico for their suitability to amplify other insect groups. The fwh1 primer sets showed the most consistent amplification in silico and in vitro , detecting 92% of the taxa present in the mock communities, and allowing clear differentiation between the three macroinvertebrate communities from the Romanian streams. In silico analysis indicates that the short primers are likely to perform well even for non-freshwater insects. Comparing the performance of the new fwh1 primer sets to a highly degenerate primer set targeting a longer fragment (BF2+BR2) revealed that detection efficiency is slightly lower for the new primer set. Nevertheless, the shorter new primer pairs might be useful for studies that have to rely on degraded or poorly conserved DNA and thus be of importance for biomonitoring, conservation biological or molecular ecological studies. Furthermore, our study highlights the need for in silico evaluation of primer sets in order to detect design errors in primers (fwhR2) and find optimal universal primer sets for the target taxa of interest.

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Comparision of two different primer sets for PCR detection of transfusion transmitted virus DNA in blood donors, hemodialysis patients and patients with thalassemia major, chronic HBV and HCV infection

Journal of Hepatology ◽

10.1016/s0168-8278(01)80596-1 ◽

2001 ◽

Vol 34 (0) ◽

pp. 163 ◽

Cited By ~ 1

Author(s):

H Verdi

Keyword(s):

Blood Donors ◽

Thalassemia Major ◽

Hemodialysis Patients ◽

Pcr Detection ◽

Hcv Infection ◽

Transmitted Virus ◽

Chronic Hbv ◽

Primer Sets ◽

Transfusion Transmitted Virus

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Optimization of whole-genome sequencing of Plasmodium falciparum from low-density dried blood spot samples

Malaria Journal ◽

10.1186/s12936-021-03630-4 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Noam B. Teyssier ◽

Anna Chen ◽

Elias M. Duarte ◽

Rene Sit ◽

Bryan Greenhouse ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Dropout Rate ◽

Dried Blood Spots ◽

High Ratio ◽

Low Density ◽

Whole Genome ◽

Cross Sectional ◽

Modest Improvement ◽

Primer Sets

Abstract Background Whole-genome sequencing (WGS) is becoming increasingly useful to study the biology, epidemiology, and ecology of malaria parasites. Despite ease of sampling, DNA extracted from dried blood spots (DBS) has a high ratio of human DNA compared to parasite DNA, which poses a challenge for downstream genetic analyses. The effects of multiple methods for DNA extraction, digestion of methylated DNA, and amplification were evaluated on the quality and fidelity of WGS data recovered from DBS. Methods Low parasite density mock DBS samples were created, extracted either with Tween-Chelex or QIAamp, treated with or without McrBC, and amplified with one of three different amplification techniques (two sWGA primer sets and one rWGA). Extraction conditions were evaluated on performance of sequencing depth, percentiles of coverage, and expected SNP concordance. Results At 100 parasites/μL, Chelex-Tween-McrBC samples had higher coverage (5 × depth = 93% genome) than QIAamp extracted samples (5 × depth = 76% genome). The two evaluated sWGA primer sets showed minor differences in overall genome coverage and SNP concordance, with a newly proposed combination of 20 primers showing a modest improvement in coverage over those previously published. Conclusions Overall, Tween-Chelex extracted samples that were treated with McrBC digestion and are amplified using 6A10AD sWGA conditions had minimal dropout rate, higher percentages of coverage at higher depth, and more accurate SNP concordance than QiaAMP extracted samples. These findings extend the results of previously reported methods, making whole genome sequencing accessible to a larger number of low density samples that are commonly encountered in cross-sectional surveys.

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Evaluation of DNA Extraction Methods Developed for Forensic and Ancient DNA Applications Using Bone Samples of Different Age

Genes ◽

10.3390/genes12020146 ◽

2021 ◽

Vol 12 (2) ◽

pp. 146

Author(s):

Catarina Xavier ◽

Mayra Eduardoff ◽

Barbara Bertoglio ◽

Christina Amory ◽

Cordula Berger ◽

...

Keyword(s):

Dna Extraction ◽

Ancient Dna ◽

Extraction Method ◽

Fragment Size ◽

Molecular Genetic ◽

Extraction Methods ◽

Degraded Dna ◽

Size Analysis ◽

Dna Fragments ◽

Dna Amount

The efficient extraction of DNA from challenging samples, such as bones, is critical for the success of downstream genotyping analysis in molecular genetic disciplines. Even though the ancient DNA community has developed several protocols targeting small DNA fragments that are typically present in decomposed or old specimens, only recently forensic geneticists have started to adopt those protocols. Here, we compare an ancient DNA extraction protocol (Dabney) with a bone extraction method (Loreille) typically used in forensics. Real-time quantitative PCR and forensically representative typing methods including fragment size analysis and sequencing were used to assess protocol performance. We used four bone samples of different age in replicates to study the effects of both extraction methods. Our results confirm Loreille’s overall increased gain of DNA when enough tissue is available and Dabney’s improved efficiency for retrieving shorter DNA fragments that is beneficial when highly degraded DNA is present. The results suggest that the choice of extraction method needs to be based on available sample, degradation state, and targeted genotyping method. We modified the Dabney protocol by pooling parallel lysates prior to purification to study gain and performance in single tube typing assays and found that up to six parallel lysates lead to an almost linear gain of extracted DNA. These data are promising for further forensic investigations as the adapted Dabney protocol combines increased sensitivity for degraded DNA with necessary total DNA amount for forensic applications.

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Development of Molecular Markers for Predicting Radish (Raphanus sativus) Flesh Color Based on Polymorphisms in the RsTT8 Gene

Plants ◽

10.3390/plants10071386 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1386

Author(s):

Soyun Kim ◽

Keunho Yun ◽

Han Yong Park ◽

Ju Young Ahn ◽

Ju Yeon Yang ◽

...

Keyword(s):

Molecular Markers ◽

Raphanus Sativus ◽

Nucleotide Polymorphisms ◽

Single Nucleotide ◽

Cosmetic Industry ◽

Natural Colorants ◽

Health Promoting ◽

Primer Sets ◽

Red Radish ◽

Simple Sequence

Red radish (Raphanus sativus L.) cultivars are a rich source of health-promoting anthocyanins and are considered a potential source of natural colorants used in the cosmetic industry. However, the development of red radish cultivars via conventional breeding is very difficult, given the unusual inheritance of the anthocyanin accumulation trait in radishes. Therefore, molecular markers linked with radish color are needed to facilitate radish breeding. Here, we characterized the RsTT8 gene isolated from four radish genotypes with different skin and flesh colors. Sequence analysis of RsTT8 revealed a large number of polymorphisms, including insertion/deletions (InDels), single nucleotide polymorphisms (SNPs), and simple sequence repeats (SSRs), between the red-fleshed and white-fleshed radish cultivars. To develop molecular markers on the basis of these polymorphisms for discriminating between radish genotypes with different colored flesh tissues, we designed four primer sets specific to the RsTT8 promoter, InDel, SSR, and WD40/acidic domain (WD/AD), and tested these primers on a diverse collection of radish lines. Except for the SSR-specific primer set, all primer sets successfully discriminated between red-fleshed and white-fleshed radish lines. Thus, we developed three molecular markers that can be efficiently used for breeding red-fleshed radish cultivars.

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