BatchPrimer3: A high throughput web application for PCR and sequencing primer design

You, Frank M.; Huo, Naxin; Gu, Yong; Luo, Ming‐Cheng; Ma, Yaqin; Dave, Hane; Lazo, Gerard R.; Dvořák, Jan; Anderson, Olin D.

doi:10.1186/1471-2105-9-253

Cited by 706 publications

(510 citation statements)

References 38 publications

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“…From among these SNPs we selected a total of 108 variable loci identified from the single copy regions, including (1) all the regions that had a variant position among the Hawaiian mints (except where every individual had an alternative allele compared to the reference sequence) and (2) additional regions that had variant positions among at least two of the Stachys species. 100 bp of flanking sequence on either side of the SNPs was retrieved from the reference genome, and PCR primers were designed using BatchPrimer3 (You et al, 2008) and examined manually for quality control. Sequences complementary to the Illumina sequencing adapters were appended to the end of each primer (see Table 9 in the companion data paper by Welch et al, 2016).…”

Section: Targeted Resequencing Of Variable Regionsmentioning

confidence: 99%

The quest to resolve recent radiations: Plastid phylogenomics of extinct and endangered Hawaiian endemic mints (Lamiaceae)

Welch

Collins

Drautz–Moses

et al. 2016

Molecular Phylogenetics and Evolution

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(2016) 'The quest to resolve recent radiations : plastid phylogenomics of extinct and endangered Hawaiian endemic mints (Lamiaceae).', Molecular phylogenetics and evolution., 99 . pp. 16-33. Further information on publisher's website: Use policyThe full-text may be used and/or reproduced, and given to third parties in any format or medium, without prior permission or charge, for personal research or study, educational, or not-for-pro t purposes provided that:• a full bibliographic reference is made to the original source • a link is made to the metadata record in DRO • the full-text is not changed in any way The full-text must not be sold in any format or medium without the formal permission of the copyright holders.Please consult the full DRO policy for further details. AbstractThe Hawaiian mints (Lamiaceae), one of the largest endemic plant lineages in the archipelago, provide an excellent system to study rapid diversification of a lineage with a remote, likely paleohybrid origin. Since their divergence from New World mints 4-5 million years ago the members of this lineage have diversified greatly and represent a remarkable array of vegetative and reproductive phenotypes. Today many members of this group are endangered or already extinct, and molecular phylogenetic work relies largely on herbarium samples collected during the last century. So far a gene-by-gene approach has been utilized, but the recent radiation of the Hawaiian mints has resulted in minimal sequence divergence and hence poor phylogenetic resolution. In our quest to trace the reticulate evolutionary history of the lineage, a resolved maternal phylogeny is necessary. We applied a high-throughput approach to sequence 12 complete or nearly complete plastid genomes from multiple Hawaiian mint species and relatives, including extinct and rare taxa. We also targeted 108 hypervariable regions from throughout the chloroplast genomes in nearly all of the remaining Hawaiian species, and relatives, using a nextgeneration amplicon sequencing approach. This procedure generated ~20Kb of sequence data for each taxon and considerably increased the total number of variable sites over previous analyses. Our results demonstrate the potential of high-throughput sequencing of historic material for evolutionary studies in rapidly evolving lineages. Our study, however, also highlights the challenges of resolving relationships within recent radiations even at the genomic level.

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Section: Targeted Resequencing Of Variable Regionsmentioning

confidence: 99%

The quest to resolve recent radiations: Plastid phylogenomics of extinct and endangered Hawaiian endemic mints (Lamiaceae)

Welch

Collins

Drautz–Moses

et al. 2016

Molecular Phylogenetics and Evolution

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“…Epoxyketone marker sequences identified within metagenomic libraries were automatically assigned to library wells by the barcode parsing functionality of the eSNaPD software package as described above. Specific primers targeting each unique EPI marker sequence were designed using BatchPrimer3 (29). To recover single clones from library wells, a serial dilution PCR strategy was used as follows: Library wells containing targets as one of ∼25,000 unique cosmids were grown overnight to confluence and diluted to a concentration of ∼4 × 10 3 CFU/mL as judged by OD 600 .…”

Section: Methodsmentioning

confidence: 99%

Multiplexed metagenome mining using short DNA sequence tags facilitates targeted discovery of epoxyketone proteasome inhibitors

Owen

Charlop–Powers²,

Smith³

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

101

107

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In molecular evolutionary analyses, short DNA sequences are used to infer phylogenetic relationships among species. Here we apply this principle to the study of bacterial biosynthesis, enabling the targeted isolation of previously unidentified natural products directly from complex metagenomes. Our approach uses short natural product sequence tags derived from conserved biosynthetic motifs to profile biosynthetic diversity in the environment and then guide the recovery of gene clusters from metagenomic libraries. The methodology is conceptually simple, requires only a small investment in sequencing, and is not computationally demanding. To demonstrate the power of this approach to natural product discovery we conducted a computational search for epoxyketone proteasome inhibitors within 185 globally distributed soil metagenomes. This led to the identification of 99 unique epoxyketone sequence tags, falling into 6 phylogenetically distinct clades. Complete gene clusters associated with nine unique tags were recovered from four saturating soil metagenomic libraries. Using heterologous expression methodologies, seven potent epoxyketone proteasome inhibitors (clarepoxcins A-E and landepoxcins A and B) were produced from these pathways, including compounds with different warhead structures and a naturally occurring halohydrin prodrug. This study provides a template for the targeted expansion of bacterially derived natural products using the global metagenome.T he advent of cost-effective high-throughput sequencing and an increasingly sophisticated understanding of bacterial secondary metabolite biosynthesis have led to two important revelations with respect to the search for new natural products: first, that the biosynthetic potential of most cultured bacteria, as judged by the number of biosynthetic gene clusters (BGCs) observed in sequenced genomes, is far greater than previously estimated (1, 2); second, that the number of bacterial species in most environments is at least 100× greater than the number of species that is readily cultured (3, 4). These observations suggest that conventional "phenotype-first" natural products isolation approaches have only examined a small fraction of earth's bacterial biosynthetic potential.There are now a number of genomic search engines available that allow researchers to rapidly scan microbial whole genome sequences for BGCs encoding new natural products (5-7). Unfortunately, the large DNA contigs that these search strategies require as input are not readily available from complex metagenomes. In response to the need for a more robust metagenomic search strategy, our group recently developed an informatics platform called eSNaPD (8, 9) (environmental Surveyor of Natural Product Diversity) with the specific aim of facilitating sequence-guided discovery of new bacterial natural products from complex metagenomes (Fig. 1).The eSNaPD software is designed to bioinformatically assess short DNA sequences that have been amplified from environmental metagenomes by degenerate PCR targeting c...

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“…The ASPE primers were designed for each marker using Primo SNP 3.4 (Chang Bioscience; www.changbioscience. com/primo/primosnp.html) or BatchPrimer3 (You et al, 2008). Cycling conditions for the ASPE reactions were 2 min at 96 1C followed by 30 cycles of 30 s at 94 1C, 1 min at 55 1C, and 2 min at 74 1C.…”

Section: Genotypingmentioning

confidence: 99%

Population structure and genetic differentiation associated with breeding history and selection in tomato (Solanum lycopersicum L.)

et al. 2010

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Tomato (Solanum lycopersicum L.) has undergone intensive selection during and following domestication. We investigated population structure and genetic differentiation within a collection of 70 tomato lines representing contemporary (processing and fresh-market) varieties, vintage varieties and landraces. The model-based Bayesian clustering software, STRUCTURE, was used to detect subpopulations. Six independent analyses were conducted using all marker data (173 markers) and five subsets of markers based on marker type (single-nucleotide polymorphisms, simple sequence repeats and insertion/deletions) and location (exon and intron sequences) within genes. All of these analyses consistently separated four groups predefined by market niche and age into distinct subpopulations. Furthermore, we detected at least two subpopulations within the processing varieties. These subpopulations correspond to historical patterns of breeding conducted for specific production environments. We found no subpopulation within fresh-market varieties, vintage varieties and landraces when using all marker data. High levels of admixture were shown in several varieties representing a transition in the demarcation between processing and fresh-market breeding. The genetic clustering detected by using the STRUCTURE software was confirmed by two statistics, pairwise F st (y) and Nei's standard genetic distance. We also identified a total of 19 loci under positive selection between processing, freshmarket and vintage germplasm by using an F st -outlier method based on the deviation from the expected distribution of F st and heterozygosity. The markers and genome locations we identified are consistent with known patterns of selection and linkage to traits that differentiate the market classes. These results demonstrate how human selection through breeding has shaped genetic variation within cultivated tomato.

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BatchPrimer3: A high throughput web application for PCR and sequencing primer design

Cited by 706 publications

References 38 publications

The quest to resolve recent radiations: Plastid phylogenomics of extinct and endangered Hawaiian endemic mints (Lamiaceae)

The quest to resolve recent radiations: Plastid phylogenomics of extinct and endangered Hawaiian endemic mints (Lamiaceae)

Multiplexed metagenome mining using short DNA sequence tags facilitates targeted discovery of epoxyketone proteasome inhibitors

Population structure and genetic differentiation associated with breeding history and selection in tomato (Solanum lycopersicum L.)

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