scholarly journals Adapterama II: universal amplicon sequencing on Illumina platforms (TaggiMatrix)

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7786 ◽  
Author(s):  
Travis C. Glenn ◽  
Todd W. Pierson ◽  
Natalia J. Bayona-Vásquez ◽  
Troy J. Kieran ◽  
Sandra L. Hoffberg ◽  
...  
Keyword(s):  
Large Scale ◽  
Amplicon Sequencing ◽  
Pcr Primers ◽  
Full Length ◽  
Link Type ◽  
Specific Pcr ◽  
Pcr Products ◽  
Next Generation Sequencing Ngs ◽  
Specific Sequences ◽  
Illumina Library

Next-generation sequencing (NGS) of amplicons is used in a wide variety of contexts. In many cases, NGS amplicon sequencing remains overly expensive and inflexible, with library preparation strategies relying upon the fusion of locus-specific primers to full-length adapter sequences with a single identifying sequence or ligating adapters onto PCR products. In Adapterama I, we presented universal stubs and primers to produce thousands of unique index combinations and a modifiable system for incorporating them into Illumina libraries. Here, we describe multiple ways to use the Adapterama system and other approaches for amplicon sequencing on Illumina instruments. In the variant we use most frequently for large-scale projects, we fuse partial adapter sequences (TruSeq or Nextera) onto the 5′ end of locus-specific PCR primers with variable-length tag sequences between the adapter and locus-specific sequences. These fusion primers can be used combinatorially to amplify samples within a 96-well plate (8 forward primers + 12 reverse primers yield 8 × 12 = 96 combinations), and the resulting amplicons can be pooled. The initial PCR products then serve as template for a second round of PCR with dual-indexed iTru or iNext primers (also used combinatorially) to make full-length libraries. The resulting quadruple-indexed amplicons have diversity at most base positions and can be pooled with any standard Illumina library for sequencing. The number of sequencing reads from the amplicon pools can be adjusted, facilitating deep sequencing when required or reducing sequencing costs per sample to an economically trivial amount when deep coverage is not needed. We demonstrate the utility and versatility of our approaches with results from six projects using different implementations of our protocols. Thus, we show that these methods facilitate amplicon library construction for Illumina instruments at reduced cost with increased flexibility. A simple web page to design fusion primers compatible with iTru primers is available at: http://baddna.uga.edu/tools-taggi.html. A fast and easy to use program to demultiplex amplicon pools with internal indexes is available at: https://github.com/lefeverde/Mr_Demuxy.

scholarly journals Adapterama II: Universal amplicon sequencing on Illumina platforms (TaggiMatrix)

2019 ◽  
Author(s):  
Travis C. Glenn ◽  
Todd W. Pierson ◽  
Natalia J. Bayona-Vásquez ◽  
Troy J. Kieran ◽  
Sandra L. Hoffberg ◽  
...  
Keyword(s):  
Large Scale ◽  
Amplicon Sequencing ◽  
Pcr Primers ◽  
Full Length ◽  
Link Type ◽  
Specific Pcr ◽  
Pcr Products ◽  
Next Generation Sequencing Ngs ◽  
Specific Sequences ◽  
Illumina Library

AbstractNext-generation sequencing (NGS) of amplicons is used in a wide variety of contexts. Most NGS amplicon sequencing remains overly expensive and inflexible, with library preparation strategies relying upon the fusion of locus-specific primers to full-length adapter sequences with a single identifying sequence or ligating adapters onto PCR products. In Adapterama I, we presented universal stubs and primers to produce thousands of unique index combinations and a modifiable system for incorporating them into Illumina libraries. Here, we describe multiple ways to use the Adapterama system and other approaches for amplicon sequencing on Illumina instruments. In the variant we use most frequently for large-scale projects, we fuse partial adapter sequences (TruSeq or Nextera) onto the 5’ end of locus-specific PCR primers with variable-length tag sequences between the adapter and locus-specific sequences. These fusion primers can be used combinatorially to amplify samples within a 96-well plate (eight forward primers + 12 reverse primers yield 8 × 12 = 96 combinations), and the resulting amplicons can be pooled. The initial PCR products then serve as template for a second round of PCR with dual-indexed iTru or iNext primers (also used combinatorially) to make full-length libraries. The resulting quadruple-indexed amplicons have diversity at most base positions and can be pooled with any standard Illumina library for sequencing. The number of sequencing reads from the amplicon pools can be adjusted, facilitating deep sequencing when required or reducing sequencing costs per sample to an economically trivial amount when deep coverage is not needed. We demonstrate the utility and versatility of our approaches with results from six projects using different implementations of our protocols. Thus, we show that these methods facilitate amplicon library construction for Illumina instruments at reduced cost with increased flexibility. A simple web page to design fusion primers compatible with iTru primers is available at: http://baddna.uga.edu/tools-taggi.html. A fast and easy to use program to demultiplex amplicon pools with internal indexes is available at: https://github.com/lefeverde/Mr_Demuxy.

scholarly journals Specific PCR detection of Peptostreptococcus magnus

2003 ◽  
Vol 52 (4) ◽  
pp. 309-313 ◽  
Author(s):  
M.P. Riggio ◽  
A. Lennon
Keyword(s):  
Pcr Primers ◽  
Oral Bacteria ◽  
Rrna Gene ◽  
Subgingival Plaque ◽  
Pcr Assay ◽  
Clinical Specimens ◽  
Specific Pcr ◽  
Wide Range ◽  
Pcr Products ◽  
Peptostreptococcus Magnus

Peptostreptococcus magnus is the most pathogenic and one of the most common Gram-positive anaerobic cocci found in human clinical specimens. The organism has been isolated in pure culture from a range of serious infections, including meningitis and endocarditis. However, isolation of Peptostreptococcus magnus from the oral cavity has rarely been attempted. Identification of Peptostreptococcus magnus in clinical specimens is reliant upon microbiological culture and biochemical methods, which often give ambiguous results. The aim of this study was to develop a PCR assay for the specific detection of Peptostreptococcus magnus in oral clinical specimens. PCR primers specific for Peptostreptococcus magnus DNA were derived by comparison of 16S rRNA gene sequences and selection of primers that demonstrated specificity at their 3′ ends for Peptostreptococcus magnus. PCR positivity for Peptostreptococcus magnus DNA was indicated by the amplification of a 553 bp product. The PCR assay was then used to attempt detection of Peptostreptococcus magnus DNA in subgingival plaque samples from adult periodontitis patients and pus aspirates from subjects with acute dento-alveolar abscesses. The PCR assay was demonstrated to be highly specific for Peptostreptococcus magnus DNA, since no PCR products were obtained when genomic DNA from a wide range of other oral bacteria, including closely related Peptostreptococcus species, was used in the PCR assay. Confirmation of specific amplification of Peptostreptococcus magnus DNA was obtained by digestion of PCR products with the restriction endonuclease RsaI, which gives a unique restriction profile for Peptostreptococcus magnus. Of the 33 subgingival plaque samples analysed, 2 (6 %) were positive for Peptostreptococcus magnus DNA. None of the 60 pus aspirates analysed was positive for Peptostreptococcus magnus DNA. It is concluded that Peptostreptococcus magnus is not a major pathogen in adult periodontitis or dento-alveolar abscesses. The PCR assay provides a more rapid, specific and sensitive alternative to conventional methods for identification of Peptostreptococcus magnus in clinical specimens.

Full-length genomic sequence of a hepatitis C virus genotype 2c isolate (BEBE1) and the 2c-specific PCR primers

1996 ◽  
Vol 141 (3-4) ◽  
pp. 701-704 ◽  
Author(s):  
H. Nakao ◽  
H. Okamoto ◽  
H. Tokita ◽  
T. Inoue ◽  
H. Iizuka ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Genomic Sequence ◽  
Pcr Primers ◽  
Full Length ◽  
Virus Genotype ◽  
Specific Pcr

scholarly journals Enigmatic Diphyllatea eukaryotes: Culturing and targeted PacBio RS amplicon sequencing reveals a higher order taxonomic diversity and global distribution

2017 ◽  
Author(s):  
J.S. Orr Russell ◽  
Zhao Sen ◽  
Klaveness Dag ◽  
Yabuki Akinori ◽  
Ikeda Keiji ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Taxonomic Diversity ◽  
Amplicon Sequencing ◽  
Pcr Primers ◽  
Higher Order ◽  
Global Distribution ◽  
Evolutionary Significance ◽  
Order Structure ◽  
Rrna Gene ◽  
Specific Pcr

AbstractDiphyllatea is an ancient and enigmatic lineage of unicellular eukaryotes that possesses morphological features common to other deeply diverging eukaryotes, such as Amoebozoa and Excavata. In reconstruction of the evolutionary processes underlying diversification and morphological innovation among eukaryotes, Diphyllatea plays a key role together with other orphan lineages. Despite being of evolutionary significance, only three species of Diphyllatea have descripted morphology, with molecular data available from fewer. The lack of data means that the actual diversity of this key lineage of eukaryotes remains unresolved. We here present a first attempt to understand the species diversity and higher order structure of the Diphyllatea phylogeny. We have cultured several new strains, described these morphologically, and amplified their rRNA. We have sampled DNA from multiple globally distributed sites, using these as templates in a Diphyllatea-specific PCR. In contrast to recent diversity studies, which use short variable gene regions, we amplify nearly the whole 18S rRNA gene, and sequence using PacBio RS II technology, to provide enough information to resolve historically ancient speciation events. Phylogenetic inference of Diphyllatea rRNA reveals three deeply branching and distinct clades of Diphyllatea, here named Diphy I – III. Diphy I and II include the generaDiphylleiaandCollodictyon. Notably, Diphy III is here shown as novel phylogenetic clade with all strains investigated having a congruent morphology toCollodictyon triciliatum(Diphy II). Altogether, Diphyllatea seems to constitute two morphotypes, a biflagellate (i.e. Diphy I) and a quadraflagellate (i.e. Diphy II and III) form, congruent with earlier descriptions ofDiphylleia and Collodictyon.Further, our targeted environmental sequencing approach, which includes specific PCR primers, reveals a wider global distribution of Diphyllatea than earlier known. Altogether, the described protocol shows the usefulness of combining long amplicon high-throughput sequencing and lineage-specific PCR approach in surveys of enigmatic eukaryote lineages.

Detection of Salmonella in Chicken Meat by Insulated Isothermal PCR

2013 ◽  
Vol 76 (8) ◽  
pp. 1322-1329 ◽  
Author(s):  
HAU-YANG TSEN ◽  
CHIA-MING SHIH ◽  
PING-HUA TENG ◽  
HSIN-YEN CHEN ◽  
CHIA-WEI LIN ◽  
...  
Keyword(s):  
Pcr Primers ◽  
Chicken Meat ◽  
Poultry Meat ◽  
Taqman Probe ◽  
Conventional Pcr ◽  
Gene Encoding ◽  
Specific Pcr ◽  
Pcr Products ◽  
Meat Samples ◽  
Insulated Isothermal Pcr

Consumption of Salmonella-contaminated foods, such as poultry and fresh eggs, is known to be one of the main causes of salmonellosis. Conventional PCR methods, including real-time PCR for rapid detection of Salmonella, in general require skilled technicians and costly instruments. A recently developed novel convective PCR, insulated isothermal PCR (iiPCR), is carried out in polycarbonate capillary tubes. In this study, we designed TaqMan probes and PCR primers based on the yrfH gene encoding a heat shock protein for the iiPCR detection of Salmonella in chicken meat samples. The TaqMan probe was labeled with 6-carboxyfluorescein and 6-carboxytetramethylrhodamine at the 5′ and 3′ ends, respectively. The PCR amplicon was 133 bp. A typical run of this iiPCR assay was completed within 1 h. Specific PCR products were obtained for 148 strains representing 49 serotypes of Salmonella tested. Under the same conditions, false-positive results were not obtained for 98 non-Salmonella strains tested, including strains of Enterobacteriaceae closely related to Salmonella. For chicken meat samples, with a 5-h enrichment step Salmonella at as low as 100 CFU/g of poultry meat could be detected. Because the amplification signals from the probes are detectable at 520 nm, identification of the PCR products by gel electrophoresis is not required. Compared with conventional PCR, the iiPCR system requires less expertise and provides an economical, reliable, and rapid tool for result interpretation. Detection results can be obtained within 8 h, including the enrichment and DNA extraction steps.

Rapid and reliable identification of rice genomes by RFLP analysis of PCR-amplified Adh genes

Genome ◽  
2001 ◽  
Vol 44 (6) ◽  
pp. 1136-1142 ◽  
Author(s):  
Song Ge ◽  
Tao Sang ◽  
Bao-rong Lu ◽  
De-yuan Hong
Keyword(s):  
Rflp Analysis ◽  
Restriction Enzymes ◽  
Pcr Primers ◽  
Specific Pcr ◽  
Adh Genes ◽  
Pcr Rflp ◽  
Restriction Sites ◽  
Pcr Products ◽  
Adh Gene ◽  
Genus Oryza

The rice genus (Oryza L.) consists of 24 species with 10 recognized genome types. With the realization of many useful genes in species of wild rice, continuous efforts have been made to understand their genomic composition and relationships. However, the identification of rice genomes has often been difficult owing to complex morphological variation and formation of allotetraploids. Here we propose a rapid and reliable method for identifying rice genomes based on the restriction sites of PCR-amplified Adh genes. The experimental procedure was as follows: (i) amplify a portion of Adh1 and Adh2 genes with the locus-specific PCR primers; (ii) digest PCR products with restriction enzymes that distinguish different genomes; and (iii) run the digested products on 1.4% agarose gel, and photograph. Using various combinations of restriction digestion of the two Adh genes, all of the rice genomes can be identified.Key words: Adh gene, genome, identification, Oryza L., PCR–RFLP.

scholarly journals Computer-Based Methods for the Mouse Full-Length cDNA Encyclopedia: Real-Time Sequence Clustering for Construction of a Nonredundant cDNA Library

2001 ◽  
Vol 11 (2) ◽  
pp. 281-289
Author(s):  
Hideaki Konno ◽  
Yoshifumi Fukunishi ◽  
Kazuhiro Shibata ◽  
Masayoshi Itoh ◽  
Piero Carninci ◽  
...  
Keyword(s):  
Cdna Library ◽  
Large Scale ◽  
Sequence Data ◽  
Full Length ◽  
Cdna Libraries ◽  
Full Length Cdna ◽  
Link Type ◽  
Computer Based ◽  
End Sequences ◽  
Press Time

We developed computer-based methods for constructing a nonredundant mouse full-length cDNA library. Our cDNA library construction process comprises assessment of library quality, sequencing the 3′ ends of inserts and clustering, and completing a re-array to generate a nonredundant library from a redundant one. After the cDNA libraries are generated, we sequence the 5′ ends of the inserts to check the quality of the library; then we determine the sequencing priority of each library. Selected libraries undergo large-scale sequencing of the 3′ ends of the inserts and clustering of the tag sequences. After clustering, the nonredundant library is constructed from the original libraries, which have redundant clones. All libraries, plates, clones, sequences, and clusters are uniquely identified, and all information is saved in the database according to this identifier. At press time, our system has been in place for the past two years; we have clustered 939,725 3′ end sequences into 127,385 groups from 227 cDNA libraries/sublibraries (seehttp://genome.gse.riken.go.jp/).[The sequence data described in this paper have been submitted to the DDBJ data library under accession nos. AV00011–AV175734, AV204013–AV382295, andBB561685–BB609425.]

AFLP-derived, Codominant Markers for Locus-specific Applications

HortScience ◽  
1998 ◽  
Vol 33 (3) ◽  
pp. 514e-514
Author(s):  
James M. Bradeen ◽  
Philipp W. Simon
Keyword(s):  
Linkage Mapping ◽  
Large Scale ◽  
Pcr Primers ◽  
Inverse Pcr ◽  
Sequence Information ◽  
Pcr Assay ◽  
Specific Primers ◽  
Simultaneous Evaluation ◽  
Feral Populations ◽  
Diversity Assessment

The amplified fragment length polymorphism (AFLP) is a powerful marker, allowing rapid and simultaneous evaluation of multiple potentially polymorphic sites. Although well-adapted to linkage mapping and diversity assessment, AFLPs are primarily dominant in nature. Dominance, relatively high cost, and technological difficulty limit use of AFLPs for marker-aided selection and other locus-specific applications. In carrot the Y2 locus conditions carotene accumulation in the root xylem. We identified AFLP fragments linked to the dominant Y2 allele and pursued conversion of those fragments to codominant, PCR-based forms useful for locus-specific applications. The short length of AFLPs (≈60 to 500 bp) precludes development of longer, more specific primers as in SCAR development. Instead, using sequence information from cloned AFLP fragments for primer design, regions outside of the original fragment were amplified by inverse PCR or ligation-mediated PCR, cloned, and sequenced. Differences in sequences associated with Y2 vs. y2 allowed development of simple PCR assays differentiating those alleles. PCR primers flanking an insertion associated with the recessive allele amplified differently sized products for the two Y2 alleles in one assay. This assay is rapid, technologically simple (requiring no radioactivity and little advanced training or equipment), reliable, inexpensive, and codominant. Our PCR assay has a variety of large scale, locus-specific applications including genotyping diverse carrot cultivars and wild and feral populations. Efforts are underway to improve upon conversion technology and to more extensively test the techniques we have developed.

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