scholarly journals Adapterama I: universal stubs and primers for 384 unique dual-indexed or 147,456 combinatorially-indexed Illumina libraries (iTru & iNext)

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7755 ◽  
Author(s):  
Travis C. Glenn ◽  
Roger A. Nilsen ◽  
Troy J. Kieran ◽  
Jon G. Sanders ◽  
Natalia J. Bayona-Vásquez ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Pcr Primers ◽  
Sequence Length ◽  
Construction Process ◽  
Start Up ◽  
Dna Libraries ◽  
Index Sequence ◽  
Primer Sets ◽  
Validation Tests ◽  
Cost Factors

Massively parallel DNA sequencing offers many benefits, but major inhibitory cost factors include: (1) start-up (i.e., purchasing initial reagents and equipment); (2) buy-in (i.e., getting the smallest possible amount of data from a run); and (3) sample preparation. Reducing sample preparation costs is commonly addressed, but start-up and buy-in costs are rarely addressed. We present dual-indexing systems to address all three of these issues. By breaking the library construction process into universal, re-usable, combinatorial components, we reduce all costs, while increasing the number of samples and the variety of library types that can be combined within runs. We accomplish this by extending the Illumina TruSeq dual-indexing approach to 768 (384 + 384) indexed primers that produce 384 unique dual-indexes or 147,456 (384 × 384) unique combinations. We maintain eight nucleotide indexes, with many that are compatible with Illumina index sequences. We synthesized these indexing primers, purifying them with only standard desalting and placing small aliquots in replicate plates. In qPCR validation tests, 206 of 208 primers tested passed (99% success). We then created hundreds of libraries in various scenarios. Our approach reduces start-up and per-sample costs by requiring only one universal adapter that works with indexed PCR primers to uniquely identify samples. Our approach reduces buy-in costs because: (1) relatively few oligonucleotides are needed to produce a large number of indexed libraries; and (2) the large number of possible primers allows researchers to use unique primer sets for different projects, which facilitates pooling of samples during sequencing. Our libraries make use of standard Illumina sequencing primers and index sequence length and are demultiplexed with standard Illumina software, thereby minimizing customization headaches. In subsequent Adapterama papers, we use these same primers with different adapter stubs to construct amplicon and restriction-site associated DNA libraries, but their use can be expanded to any type of library sequenced on Illumina platforms.

scholarly journals Adapterama I: Universal stubs and primers for 384 unique dual-indexed or 147,456 combinatorially-indexed Illumina libraries (iTru & iNext)

2016 ◽  
Author(s):  
Travis C. Glenn ◽  
Roger A. Nilsen ◽  
Troy J. Kieran ◽  
Jon G. Sanders ◽  
Natalia J. Bayona-Vásquez ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Pcr Primers ◽  
Sequence Length ◽  
Start Up ◽  
Dna Libraries ◽  
Index Sequence ◽  
Reducing Costs ◽  
Primer Sets ◽  
Major Factors ◽  
Validation Tests

AbstractNext-generation DNA sequencing (NGS) offers many benefits, but major factors limiting NGS include reducing costs of: 1) start-up (i.e., doing NGS for the first time); 2) buy-in (i.e., getting the smallest possible amount of data from a run); and 3) sample preparation. Reducing sample preparation costs is commonly addressed, but start-up and buy-in costs are rarely addressed. We present dual-indexing systems to address all three of these issues. By breaking the library construction process into universal, re-usable, combinatorial components, we reduce all costs, while increasing the number of samples and the variety of library types that can be combined within runs. We accomplish this by extending the Illumina TruSeq dual-indexing approach to 768 (384 + 384) indexed primers that produce 384 unique dual-indexes or 147,456 (384 × 384) unique combinations. We maintain eight nucleotide indexes, with many that are compatible with Illumina index sequences. We synthesized these indexing primers, purifying them with only standard desalting and placing small aliquots in replicate plates. In qPCR validation tests, 206 of 208 primers tested passed (99% success). We then created hundreds of libraries in various scenarios. Our approach reduces start-up and per-sample costs by requiring only one universal adapter that works with indexed PCR primers to uniquely identify samples. Our approach reduces buy-in costs because: 1) relatively few oligonucleotides are needed to produce a large number of indexed libraries; and 2) the large number of possible primers allows researchers to use unique primer sets for different projects, which facilitates pooling of samples during sequencing. Our libraries make use of standard Illumina sequencing primers and index sequence length and are demultiplexed with standard Illumina software, thereby minimizing customization headaches. In subsequent Adapterama papers, we use these same primers with different adapter stubs to construct amplicon and restriction-site associated DNA libraries, but their use can be expanded to any type of library sequenced on Illumina platforms.

scholarly journals A strategy to detect and isolate an intron-containing gene in the presence of multiple processed pseudogenes

1989 ◽  
Vol 86 (17) ◽  
pp. 6691-6695 ◽  
Author(s):  
B Davies ◽  
S Feo ◽  
E Heard ◽  
M Fried
Keyword(s):  
Ribosomal Protein ◽  
Recombinant Dna ◽  
Single Gene ◽  
Ribosomal Protein Gene ◽  
Pcr Primers ◽  
Pcr Product ◽  
Dna Libraries ◽  
Processed Pseudogenes ◽  
Polymerase Chain ◽  
Genomic Dna Sequence

We have devised a strategy that utilizes the polymerase chain reaction (PCR) for the detection and isolation of intron-containing genes in the presence of an abundance of processed pseudogenes. The method depends on the genomic DNA sequence between the PCR primers spanning at least one intron in the gene of interest, resulting in the generation of a larger intron-containing PCR product in addition to the smaller PCR product amplified from the intronless pseudogenes. A unique intron probe isolated from the larger PCR product is used for the detection of intron-containing clones from recombinant DNA libraries that also contain pseudogene clones. This method has been used successfully for the selective isolation of an intron-containing rat L19 ribosomal protein gene in the presence of multiple pseudogenes. Analysis of a number of mammalian ribosomal protein multigene families by PCR indicates that they all contain only a single gene with introns.

scholarly journals Design and Evaluation of PCR Primers for Analysis of Bacterial Populations in Wine by Denaturing Gradient Gel Electrophoresis

2003 ◽  
Vol 69 (11) ◽  
pp. 6801-6807 ◽  
Author(s):  
Isabel Lopez ◽  
Fernanda Ruiz-Larrea ◽  
Luca Cocolin ◽  
Erica Orr ◽  
Trevor Phister ◽  
...  
Keyword(s):  
Lactic Acid ◽  
16S Rdna ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Bacterial Species ◽  
Pcr Primers ◽  
Fungal Species ◽  
Bacterial Populations ◽  
Gradient Gel Electrophoresis ◽  
Primer Sets

ABSTRACT Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified ribosomal DNA (rDNA) is routinely used to compare levels of diversity of microbial communities and to monitor population dynamics. While using PCR-DGGE to examine the bacteria in wine fermentations, we noted that several commonly used PCR primers for amplifying bacterial 16S rDNA also coamplified yeast, fungal, or plant DNA present in samples. Unfortunately, amplification of nonbacterial DNA can result in a masking of bacterial populations in DGGE profiles. To surmount this problem, we developed two new primer sets for specific amplification of bacterial 16S rDNA in wine fermentation samples without amplification of eukaryotic DNA. One primer set, termed WLAB1 and WLAB2, amplified lactic acid bacteria, while another, termed WBAC1 and WBAC2, amplified both lactic acid bacterial and acetic acid bacterial populations found in wine. Primer specificity and efficacy were examined with DNA isolated from numerous bacterial, yeast, and fungal species commonly found in wine and must samples. Importantly, both primer sets effectively distinguished bacterial species in wine containing mixtures of yeast and bacteria.

scholarly journals Development and Validation of Broad-Range Qualitative and Clade-Specific Quantitative Molecular Probes for Assessing Mercury Methylation in the Environment

2016 ◽  
Vol 82 (19) ◽  
pp. 6068-6078 ◽  
Author(s):  
Geoff A. Christensen ◽  
Ann M. Wymore ◽  
Andrew J. King ◽  
Mircea Podar ◽  
Richard A. Hurt ◽  
...  
Keyword(s):  
Pcr Primers ◽  
Molecular Probes ◽  
Accurate Identification ◽  
Content Type ◽  
Anaerobic Microorganisms ◽  
Risk Environments ◽  
Pure Cultures ◽  
Primer Sets ◽  
The Individual ◽  
Development And Validation

ABSTRACTTwo genes,hgcAandhgcB, are essential for microbial mercury (Hg) methylation. Detection and estimation of their abundance, in conjunction with Hg concentration, bioavailability, and biogeochemistry, are critical in determining potential hot spots of methylmercury (MeHg) generation in at-risk environments. We developed broad-range degenerate PCR primers spanning knownhgcABgenes to determine the presence of both genes in diverse environments. These primers were tested against an extensive set of pure cultures with published genomes, including 13Deltaproteobacteria, nineFirmicutes, and nine methanogenicArchaeagenomes. A distinct PCR product at the expected size was confirmed for allhgcAB+strains tested via Sanger sequencing. Additionally, we developed clade-specific degenerate quantitative PCR (qPCR) primers that targetedhgcAfor each of the three dominant Hg-methylating clades. The clade-specific qPCR primers amplifiedhgcAfrom 64%, 88%, and 86% of tested pure cultures ofDeltaproteobacteria,Firmicutes, andArchaea, respectively, and were highly specific for each clade. Amplification efficiencies and detection limits were quantified for each organism. Primer sensitivity varied among species based on sequence conservation. Finally, to begin to evaluate the utility of our primer sets in nature, we testedhgcAandhgcABrecovery from pure cultures spiked into sand and soil. These novel quantitative molecular tools designed in this study will allow for more accurate identification and quantification of the individual Hg-methylating groups of microorganisms in the environment. The resulting data will be essential in developing accurate and robust predictive models of Hg methylation potential, ideally integrating the geochemistry of Hg methylation to the microbiology and genetics ofhgcAB.IMPORTANCEThe neurotoxin methylmercury (MeHg) poses a serious risk to human health. MeHg production in nature is associated with anaerobic microorganisms. The recent discovery of the Hg-methylating gene pair,hgcAandhgcB, has allowed us to design and optimize molecular probes against these genes within the genomic DNA for microorganisms known to methylate Hg. The protocols designed in this study allow for both qualitative and quantitative assessments of pure-culture or environmental samples. With these protocols in hand, we can begin to study the distribution of Hg-methylating organisms in nature via a cultivation-independent strategy.

scholarly journals openPrimeR for multiplex amplification of highly diverse templates

2019 ◽  
Author(s):  
Christoph Kreer ◽  
Matthias Döring ◽  
Nathalie Lehnen ◽  
Meryem S. Ercanoglu ◽  
Lutz Gieselmann ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Gene Segment ◽  
Pcr Primers ◽  
Proof Of Concept ◽  
Minimal Set ◽  
Multiplex Amplification ◽  
Variable Gene ◽  
Primer Sets ◽  
Correct Understanding ◽  
Intuitive Interface

AbstractTo study the diversity of immune receptors and pathogens, multiplex PCR has become a central approach in research and diagnostics. However, insufficient primer design against highly diverse templates often prevents amplification and therefore limits the correct understanding of biological processes. Here, we present openPrimeR, an R-based tool for evaluating and designing multiplex PCR primers. openPrimeR provides a functional and intuitive interface and uses either a greedy algorithm or an integer linear program to compute the minimal set of primers that performs full target coverage. As proof of concept, we used openPrimeR to find optimal primer sets for the amplification of highly mutated immunoglobulins. Comprehensive analyses on specifically generated immunoglobulin variable gene segment libraries resulted in the composition of highly effective primer sets (oPR-IGHV, oPR-IGKV and oPR-IGLV) that demonstrated to be particularly suitable for the isolation of novel human antibodies.

scholarly journals Evaluation of PCR Primers for Early Diagnosis of Cytomegalovirus Infection following Liver Transplantation

1998 ◽  
Vol 36 (2) ◽  
pp. 526-530 ◽  
Author(s):  
Julio C. Mendez ◽  
Mark J. Espy ◽  
Thomas F. Smith ◽  
Jennie A. Wilson ◽  
Carlos V. Paya
Keyword(s):  
Early Detection ◽  
Liver Transplant ◽  
Pcr Primers ◽  
Immediate Early ◽  
Transplant Recipients ◽  
Blood Leukocytes ◽  
Symptomatic Infection ◽  
Primer Sets ◽  
Early Replication ◽  
Liver Transplant Recipients

The availability of microbiologic methods that detect early replication of cytomegalovirus (CMV) posttransplantation will enhance the process of initiating preemptive antiviral therapy prior to the appearance of CMV disease. Using PCR techniques we sought to determine which region of the CMV genome present in peripheral blood leukocytes (PBLs) or serum provides the highest sensitivity for the detection of CMV posttransplantation. Blood samples were prospectively collected weekly for at least 8 weeks from a cohort of 21 consecutive liver transplant recipients not receiving anti-CMV prophylaxis. Results of PCR assays were correlated with recovery of CMV in cell cultures and histopathological findings from biopsy specimens of infected organs to assess clinical symptomatic infection. Of 148 specimens, primer pairs directed to the HindIII-X fragment region of CMV detected target DNA with a 94% sensitivity, compared to an 87% sensitivity with primer pairs directed to EcoRI fragment D, 32% sensitivity with primer pairs directed to the immediate-early antigen 1 gene (IEA1 gene), and 20% sensitivity with primer pairs directed to the major immediate-early (MIE) gene. The performance characteristics in terms of the sensitivity of primers for amplifying CMV DNA associated with symptomatic infection ranged from 100% (HindIII-X) to 20% (MIE gene); however, specificity was inversely related (HindIII-X, 45%; MIE gene, 91%) to primers directed to these gene targets. When HindIII-X andEcoRI-D primer sets were used, CMV DNA from PBLs was a more sensitive target than CMV DNA from serum for the early detection of symptomatic CMV infection (17 versus 12 days). Importantly, CMV DNA was not detected in five patients with no evidence of this viral infection. In conclusion, primers directed to the HindIII-X fragment region were the most optimal for the early detection of CMV DNA in PBLs and sera from symptomatic liver transplant recipients.

scholarly journals Semi-Automated Library Preparation for High-Throughput DNA Sequencing Platforms

2010 ◽  
Vol 2010 ◽  
pp. 1-8 ◽  
Author(s):  
Eveline Farias-Hesson ◽  
Jonathan Erikson ◽  
Alexander Atkins ◽  
Peidong Shen ◽  
Ronald W. Davis ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Dna Sequencing ◽  
High Throughput ◽  
Magnetic Beads ◽  
Dna Barcode ◽  
Cost Effective ◽  
Dna Libraries ◽  
Emulsion Pcr ◽  
High Throughput Dna Sequencing ◽  
Sequencing Platforms

Next-generation sequencing platforms are powerful technologies, providing gigabases of genetic information in a single run. An important prerequisite for high-throughput DNA sequencing is the development of robust and cost-effective preprocessing protocols for DNA sample library construction. Here we report the development of a semi-automated sample preparation protocol to produce adaptor-ligated fragment libraries. Using a liquid-handling robot in conjunction with Carboxy Terminated Magnetic Beads, we labeled each library sample using a unique 6 bp DNA barcode, which allowed multiplex sample processing and sequencing of 32 libraries in a single run using Applied Biosystems' SOLiD sequencer. We applied our semi-automated pipeline to targeted medical resequencing of nuclear candidate genes in individuals affected by mitochondrial disorders. This novel method is capable of preparing as much as 32 DNA libraries in 2.01 days (8-hour workday) for emulsion PCR/high throughput DNA sequencing, increasing sample preparation production by 8-fold.

An integrated SSR and RFLP linkage map of Sorghum bicolor (L.) Moench

Genome ◽  
2000 ◽  
Vol 43 (6) ◽  
pp. 988-1002 ◽  
Author(s):  
Dinakar Bhattramakki ◽  
Jianmin Dong ◽  
Ashok K Chhabra ◽  
Gary E Hart
Keyword(s):  
Sorghum Bicolor ◽  
Linkage Map ◽  
Dna Sequences ◽  
Artificial Chromosome ◽  
Polymorphic Loci ◽  
Dna Libraries ◽  
Ssr Loci ◽  
Bac Libraries ◽  
Primer Sets ◽  
Sorghum Bicolor L

We report the development, testing, and use (for genetic mapping) of a large number of polymerase chain reaction (PCR) primer sets that amplify DNA simple sequence repeat (SSR) loci of Sorghum bicolor (L.) Moench. Most of the primer sets were developed from clones isolated from two sorghum bacterial artificial chromosome (BAC) libraries and three enriched sorghum genomic-DNA (gDNA) libraries. A few were developed from sorghum DNA sequences present in public databases. The libraries were probed with radiolabeled di- and trinucleotide oligomers, the BAC libraries with four and six oligomers, respectively, and the enriched gDNA libraries with four and three oligomers, respectively. Both types of libraries were markedly enriched for SSRs relative to a size-fractionated gDNA library studied earlier. However, only 2% of the sequenced clones obtained from the size-fractionated gDNA library lacked a SSR, whereas 13% and 17% of the sequenced clones obtained from the BAC and enriched gDNA libraries, respectively, lacked a SSR. Primer sets were produced for 313 SSR loci. Two-hundred sixty-six (85%) of the loci were amplified and 165 (53%) of the loci were found to be polymorphic in a population composed of 18 diverse sorghum lines. (AG/TC)n and (AC/TG)n repeats comprised 91% of the dinucleotide SSRs and 52% of all of the SSRs at the polymorphic loci, whereas four types of repeats comprised 66% of the trinucleotide SSRs at the loci. Primer sequences are reported for the 165 polymorphic loci and for eight monomorphic loci that have a high degree of homology to genes. Also reported are the genetic map locations of 113 novel SSR loci (including four SSR-containing gene loci) and a linkage map composed of 147 SSR loci and 323 RFLP (restriction fragment length polymorphism) loci. The number of SSR loci per linkage group ranges from 8 to 30. The SSR loci are distributed relatively evenly throughout approximately 75% of the 1406-cM linkage map, but segments of five linkage groups comprising about 25% of the map either lack or contain few SSR loci. Mapping of SSR loci isolated from BAC clones located to these segments is likely to be the most efficient method for placing SSR loci in the segments.Key words: DNA libraries, linkage mapping, Sorghum bicolor, SSRs.

scholarly journals Diet-Dependent Shifts in the Bacterial Population of the Rumen Revealed with Real-Time PCR

2001 ◽  
Vol 67 (6) ◽  
pp. 2766-2774 ◽  
Author(s):  
K. Tajima ◽  
R. I. Aminov ◽  
T. Nagamine ◽  
H. Matsui ◽  
M. Nakamura ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Transition Period ◽  
Fold Increase ◽  
Pcr Primers ◽  
The Real ◽  
Prevotella Ruminicola ◽  
Fold Reduction ◽  
Dna Libraries ◽  
Diet Switch

ABSTRACT A set of PCR primers was designed and validated for specific detection and quantification of Prevotella ruminicola,Prevotella albensis, Prevotella bryantii,Fibrobacter succinogenes, Selenomonas ruminantium-Mitsuokella multiacida, Streptococcus bovis, Ruminococcus flavefaciens,Ruminobacter amylophilus, Eubacterium ruminantium, Treponema bryantii,Succinivibrio dextrinosolvens, and Anaerovibrio lipolytica. By using these primers and the real-time PCR technique, the corresponding species in the rumens of cows for which the diet was switched from hay to grain were quantitatively monitored. The dynamics of two fibrolytic bacteria, F. succinogenesand R. flavefaciens, were in agreement with those of earlier, culture-based experiments. The quantity of F. succinogenes DNA, predominant in animals on the hay diet, fell 20-fold on the third day of the switch to a grain diet and further declined on day 28, with a 57-fold reduction in DNA. TheR. flavefaciens DNA concentration on day 3 declined to approximately 10% of its initial value in animals on the hay diet and remained at this level on day 28. During the transition period (day 3), the quantities of two ruminal prevotella DNAs increased considerably: that of P. ruminicola increased 7-fold and that ofP. bryantii increased 263-fold. On day 28, the quantity of P. ruminicola DNA decreased 3-fold, while P. bryantii DNA was still elevated 10-fold in comparison with the level found in animals on the initial hay diet. The DNA specific for another xylanolytic bacterium, E. ruminantium, dropped 14-fold during the diet switch and was maintained at this level on day 28. The concentration of a rumen spirochete, T. bryantii, decreased less profoundly and stabilized with a sevenfold decline by day 28. The variations in A. lipolytica DNA were not statistically significant. After an initial slight increase in S. dextrinosolvens DNA on day 3, this DNA was not detected at the end of the experiment. S. bovis DNA displayed a 67-fold increase during the transition period on day 3. However, on day 28, it actually declined in comparison with the level in animals on the hay ration. The amount of S. ruminantium-M. multiacida DNA also increased eightfold following the diet switch, but stabilized with only a twofold increase on day 28. The real-time PCR technique also uncovered differential amplification of rumen bacterial templates with the set of universal bacterial primers. This observation may explain why some predominant rumen bacteria have not been detected in PCR-generated 16S ribosomal DNA libraries.

scholarly journals Detection of Enterotoxic Bacillus cereus andBacillus thuringiensis Strains by PCR Analysis

2001 ◽  
Vol 67 (1) ◽  
pp. 185-189 ◽  
Author(s):  
Bjarne Munk Hansen ◽  
Niels Bohse Hendriksen
Keyword(s):  
Bacillus Cereus ◽  
Protein Complexes ◽  
Pcr Primers ◽  
Gastrointestinal Diseases ◽  
23S Rrna ◽  
Pcr Analysis ◽  
Rrna Gene ◽  
Internal Transcribed Spacer Region ◽  
Primer Sets ◽  
Different Strains

ABSTRACT Many strains of Bacillus cereus cause gastrointestinal diseases, and the closely related insect pathogen B. thuringiensis has also been involved in outbreaks of diarrhea. The diarrheal types of diseases are attributed to enterotoxins. Two different enterotoxic protein complexes, hemolysin BL (HBL) and nonhemolytic enterotoxin (NHE), and an enterotoxic protein, enterotoxin T, have been characterized, and the genes have been sequenced. PCR primers for the detection of these genes were deduced and used to detect the genes in 22 B. cereus and 41 B. thuringiensis strains. At least one gene of each of the two protein complexes HBL and NHE was detected in all of the B. thuringiensis strains, while six B. cereus strains were devoid of all three HBL genes, three lacked at least two of the three NHE genes, and one lacked all three. Five different sets of primers were used for detection of the gene (bceT) encoding enterotoxin T. The results obtained with these primer sets indicate that bceT is widely distributed among B. cereusand B. thuringiensis strains and that the gene varies in sequence among different strains. PCR with the two primer sets BCET1-BCET3 and BCET1-BCET4 unambiguously detected the bceTgene, as confirmed by Southern analysis. The occurrence of the genes within the two complexes is significantly associated, while neither the occurrence of the two complexes nor the occurrence of thebceT gene is significantly associated in the 63 strains. We suggest an approach for detection of enterotoxin-encoding genes inB. cereus and B. thuringiensis based on PCR analysis with the six primer sets for the detection of genes in the HBL and NHE operons and with the BCET1, BCET3, and BCET4 primers for the detection of bceT. PCR analysis of the 16S-23S rRNA gene internal transcribed spacer region revealed identical patterns for all strains studied.

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