MPprimer: a program for reliable multiplex PCR primer design

Shen, Zhiyong; Qu, Wubin; Wang, Wen; Lu, Yiming; Wu, Yonghong; Li, Zhifeng; Hang, Xingyi; Wang, Xiaolei; Zhao, Dongsheng; Zhang, Chenggang

doi:10.1186/1471-2105-11-143

Cited by 148 publications

(118 citation statements)

References 22 publications

(45 reference statements)

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“…It is possible that the high degeneracy of the forward primers in combination with low diversity nucleotides at the primer's 3 ′ -end (e.g., C[cta]TT[tc]CC in BF2) makes this effect particularly pronounced. Therefore, we recommend designing primers with two unique nucleotides at the 3 ′ -end e.g., CG and additionally considering common primer design guidelines (Kwok et al, 1994;Mülhardt, 2008;Shen et al, 2010). The effect of this minimal shifting, shortens read length by 1-2 bp while having no effect on the detection of taxa (OTUs will still match the same reference taxon, regardless of 1-2 bp being clipped from the sequence).…”

Section: Discussion Amplification Success Of Mock Communitiesmentioning

confidence: 99%

“…While we can recommend using the BF2 + BR2 or BF2 + BR1 primer set for targeting freshwater taxa with DNA metabarcoding, we explicitly express that for routine monitoring further improved primers would be desirable. This can be archived by testing additional degenerated primer pairs or develop multiplex primer sets (targeting the same or similar regions), while the latter have the disadvantage of adding additional laboratory costs (Mülhardt, 2008;Shen et al, 2010;Hajibabaei et al, 2012;Gibson et al, 2014).…”

Section: Discussion Amplification Success Of Mock Communitiesmentioning

confidence: 99%

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Validation and Development of COI Metabarcoding Primers for Freshwater Macroinvertebrate Bioassessment

Elbrecht

Leese

2017

Front. Environ. Sci.

224

232

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A central challenge in the present era of biodiversity loss is to assess and manage human impacts on freshwater ecosystems. Macroinvertebrates are an important group for such bioassessments as many taxa show specific responses to environmental conditions. However, generating accurate macroinvertebrate inventories based on primarily larval morphology is difficult and error-prone. Here, DNA metabarcoding provides new opportunities. Its potential to accurately identify invertebrates in bulk samples to the species level has been demonstrated in several case studies. However, DNA based identification is often limited by primer bias, potentially leading to taxa in the sample remaining undetected. Thus, the success of DNA metabarcoding as an emerging technique for bioassessment critically relies on carefully evaluated primers. We used the R package PrimerMiner to obtain and process cytochrome c oxidase I (COI) sequence data for the 15 globally most relevant freshwater invertebrate groups for stream assessment. Using these sequence alignments, we developed four primer combinations optimized for freshwater macroinvertebrates. All primers were evaluated by sequencing 10 mock community samples, each consisting of 52 freshwater invertebrate taxa. Additionally, popular metabarcoding primers from the literature and the developed primers were tested in silico against these 15 relevant invertebrate groups. The developed primers varied in amplification efficiency and the number of detected taxa, yet, all detected more taxa than standard "Folmer" barcoding primers. Two new primer combinations showed even more consistent amplification than a previously tested ribosomal marker (16S) and detected all 42 insect taxa present in the mock community samples. In silico evaluation revealed critical design flaws in some commonly used primers from the literature. We demonstrate a reliable strategy to develop optimized primers using the tool PrimerMiner. The developed primers detected almost all taxa present in the mock samples, and we argue that high base degeneracy is necessary to decrease primer bias as confirmed by experimental results and in silico primer evaluation. We further demonstrate that some primers currently Elbrecht and Leese Freshwater Primer Development and Evaluation used in metabarcoding studies may not be suitable for amplification of freshwater macroinvertebrates. Therefore, careful primer evaluation and more region/ecosystem specific primers are needed before DNA metabarcoding can be used for routine bioassessment of freshwater ecosystems.

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Section: Discussion Amplification Success Of Mock Communitiesmentioning

confidence: 99%

Section: Discussion Amplification Success Of Mock Communitiesmentioning

confidence: 99%

Validation and Development of COI Metabarcoding Primers for Freshwater Macroinvertebrate Bioassessment

Elbrecht

Leese

2017

Front. Environ. Sci.

224

232

View full text Add to dashboard Cite

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“…In the process of primer design, ΔG is one of most critical factors used to determine the presence of dimers (Shen et al 2010). Primer or primer pairs calculated by different algorithms showed different ΔG value.…”

Section: Conclusion and Discussionmentioning

confidence: 99%

miPrimer: an empirical-based qPCR primer design method for small noncoding microRNA

Kang¹,

Hsieh²,

Feng³

et al. 2017

RNA

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MicroRNA (miRNAs) are 18-25 nucleotides highly conserved, non-coding RNAs involved in gene regulations. Because of miRNAs' short length, the design of miRNA primers for PCR amplification remains a significant challenge. Adding to the challenge are miRNAs similar in sequences and miRNA family members which often only differ in sequences by one nucleotide.Here, we described a novel empirical-based method, miPrimer, which greatly reduces primer dimerization and increases primer specificity by factoring various intrinsic primer properties and employing four primer design strategies. The resulting primer pairs displayed an acceptable qPCR efficiency between 90% ~ 110%. When tested on miRNA families, miPrimer-designed primers are capable of discriminating members of miRNA families as validated by qPCR assay using Quark Biosciences' platform. Of the 120 miRNA primer pairs tested, 95.6% and 93.3%were successful in amplifying specifically non-family and family miRNA members, respectively, after only one design trial. In summary, miPrimer provides a cost-effective and valuable tool for designing miRNA primers.

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“…Subsequently, 1 lg of RNA was converted to cDNA using the Rever Tra Ace kit (Toyobo, Tokyo, Japan). Multiplex PCR primers were designed using the MPprimer software (http://biocompute.bmi.ac.cn/MPprimer/) (52). For real time PCR, the cDNA was amplified using an ABI Prism 7900HT sequencing detection system with SYBR green polymerase chain reaction master mix (Applied Biosystems).…”

Section: Glb-13 Mrna Expressionmentioning

confidence: 99%

GLB‐13 is associated with oxidative stress resistance in caenorhabditis elegans

Ren

Han

et al. 2013

IUBMB Life

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Globins constitute a superfamily of heme-binding proteins that is widely present in many species. There are 33 putative globins in the genome of Caenorhabditis elegans, where glb-13 is a homolog of neuroglobin (Ngb) based on sequence analysis and specific expression in neurons. Here we examined whether glb-13 as well as Ngb is also associated with resistance to reactive oxygen species (ROS) induced by paraquat. Our results showed that the mRNA level of glb-13 was significantly upregulated after paraquat exposure. Expression of a green fluorescent protein (GFP) reporter gene directed by the glb-13 promoter was increased by paraquat exposure. The mutant C. elegans strain glb-13(tm2825) was sensitive to paraquat-induced oxidative stress. Overexpression of human Ngb (hNgb) in C. elegans neuronal cells can rescue the paraquat sensitive phenotype of the mutant strain. glb-13 mutation or hNgb overexpression did not affect the expression of antioxidant enzymes such as superoxide dismutase (SOD). To examine the ROS-scavenging capabilities of hNgb and glb-13, we further examined the level of ROS in glb-13 mutant and hNgb transgenic (hNgb-Tg) worms. There was no statistical difference in ROS levels in the untreated controls; however in paraquattreated worms, the ROS level was statistically repressed in the hNgb-Tg relative to enhanced green fluorescent protein (EGFP)-Tg worms or wildtype animals. Additionally, the ROS level of glb-13 mutant was statistically higher than the wildtype animals. Furthermore, hNgb overexpression diminished the ROS level of glb-13 mutant. In conclusion, hNgb can rescue the ROS sensitive phenotype of the glb-13 mutant strain. The protein GLB-13 seems to have an hNgb-like function, suggesting the importance of the globin protein family in maintaining the homeostasis of ROS signals. Our data provided evidence for the first time that glb-13 is associated with the resistance against oxidative stress-induced toxicity.

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MPprimer: a program for reliable multiplex PCR primer design

Cited by 148 publications

References 22 publications

Validation and Development of COI Metabarcoding Primers for Freshwater Macroinvertebrate Bioassessment

Validation and Development of COI Metabarcoding Primers for Freshwater Macroinvertebrate Bioassessment

miPrimer: an empirical-based qPCR primer design method for small noncoding microRNA

GLB‐13 is associated with oxidative stress resistance in caenorhabditis elegans

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