Palindromic Sequence-Targeted (PST) PCR, Version 2: An Advanced Method for High-Throughput Targeted Gene Characterization and Transposon Display

Genome walking (GW), a strategy for capturing previously unsequenced DNA fragments that are in proximity to a known sequence tag, is currently predominantly based on PCR. Recently developed PCR-based methods allow for combining of sequence-specific primers with designed capturing primers capable of annealing to unknown DNA targets, thereby offering the rapidity and effectiveness of PCR. This study presents a methodological improvement to the previously described GW technique known as palindromic sequence-targeted PCR (PST-PCR). Like PST-PCR, this new method (called PST-PCR v.2) relies on targeting of capturing primers to palindromic sequences arbitrarily present in natural DNA templates. PST-PCR v.2 consists of two rounds of PCR. The first round uses a combination of one sequence-specific primer with one capturing (PST) primer. The second round uses a combination of a single (preferred) or two universal primers; one anneals to a 5′ tail attached to the sequence-specific primer and the other anneals to a different 5′ tail attached to the PST primer. The key advantage of PST-PCR v.2 is the convenience of using a single universal primer with invariable sequences in GW processes involving various templates. The entire procedure takes approximately 2–3 h to produce the amplified PCR fragment, which contains a portion of a template flanked by the sequence-specific and capturing primers. PST-PCR v.2 is highly suitable for simultaneous work with multiple samples. For this reason, PST-PCR v.2 can be applied beyond the classical task of GW for studies in population genetics, in which PST-PCR v.2 is a preferred alternative to amplified fragment length polymorphism (AFLP) or next-generation sequencing. Furthermore, the conditions for PST-PCR v.2 are easier to optimize, as only one sequence-specific primer is used. This reduces non-specific random amplified polymorphic DNA (RAPD)-like amplification and formation of non-templated amplification. Importantly, akin to the previous version, PST-PCR v.2 is not sensitive to template DNA sequence complexity or quality. This study illustrates the utility of PST-PCR v.2 for transposon display (TD), which is a method to characterize inter- or intra-specific variability related to transposon integration sites. The Ac transposon sequence in the maize (Zea mays) genome was used as a sequence tag during the TD procedure to characterize the Ac integration sites.

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Specific Detection of Fasciola hepatica and F. gigantica in Infected Domesticated Animals Using High-Resolution Melting Analysis (HRM)

Iranian Journal of Public Health ◽

10.18502/ijph.v49i3.3149 ◽

2020 ◽

Author(s):

Zeinab MOGHADAMIZAD ◽

Ahmad HOSSEINI-SAFA ◽

Mehdi MOHEBALI ◽

Peyman HEYDARIAN ◽

Mojgan ARYAEIPOUR ◽

...

Keyword(s):

High Resolution ◽

High Resolution Melting ◽

Fasciola Hepatica ◽

High Resolution Melting Analysis ◽

Universal Primers ◽

Specific Primer ◽

Universal Primer ◽

Melting Analysis ◽

Liver Flukes ◽

Fasciola Spp

Background: It is difficult to make an exact morphological distinction between Fasciola hepatica and Fasciola gigantica. We used High Resolution Melting analysis (HRM) method to differentiate the F. hepatica species from F. gigantica in order to differentiate them. Methods: Overall, 80 adult liver flukes were collected from infected slaughtered animals including cattle, sheep and goats from Lorestan Province, western Iran from Sep 2015 to Aug 2017. Genomic DNA was extracted using commercial DNA extraction kit. The multilocus sequences of mDNA including COX1, COX3 and ND6 were amplified employing real-time PCR & HRM analysis. Specific and universal primer pairs were designed for differentiation Fasciola spp. Results: Universal primers cannot be used to distinguish between these two species, but in the contrary, specific primer pairs of each species could differentiate them properly. Molecular identification using specific primer pairs were consistent. Conclusion: HRM is a simple, fast and reliable method for detecting and differentiating F. hepatica from F. gigantica and can be used for diagnostic and epidemiological purposes.

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Development of PCR Assays for the Identification of Species and Pathotypes of Elsinoë Causing Scab on Citrus

Plant Disease ◽

10.1094/pdis-91-7-0865 ◽

2007 ◽

Vol 91 (7) ◽

pp. 865-870 ◽

Cited By ~ 16

Author(s):

J. W. Hyun ◽

N. A. Peres ◽

S.-Y. Yi ◽

L. W. Timmer ◽

K. S. Kim ◽

...

Keyword(s):

Internal Transcribed Spacer ◽

Sweet Orange ◽

Random Amplified Polymorphic Dna ◽

Its Region ◽

The United States ◽

Specific Primer ◽

Identification Of Species ◽

Orange Fruit ◽

Pcr Assays ◽

Primer Sets

Two scab pathogens of citrus, Elsinoë fawcettii and E. australis, cause citrus scab and sweet orange scab, respectively, and pathotypes of each species have been described. The two species cannot be readily distinguished by morphological or cultural characteristics and can be distinguished only by host range and the sequence of the internal transcribed spacer (ITS) region. In this study, random amplified polymorphic DNA (RAPD) assays clearly distinguished E. fawcettii and E. australis, and the sweet orange and natsudaidai pathotypes within E. australis also could be differentiated. We developed specific primer sets, Efaw-1 for E. fawcettii; Eaut-1, Eaut-2, Eaut-3, and Eaut-4 for E. australis; and EaNat-1 and EaNat-2 for the natsudaidai pathotype within E. australis using RAPD products unique to each species or pathotype. Other primer sets, Efaw-2 and Eaut-5, which were specific for E. fawcettii and E. australis, respectively, were designed from previously determined ITS sequences. The Efaw-1 and Efaw-2 primer sets successfully identified E. fawcettii isolates from Korea, Australia, and the United States (Florida) and the Eaut-1 to Eaut-5 primer sets identified both the sweet orange pathotype isolates of E. australis from Argentina and the natsudaidai pathotype isolates from Korea. The EaNat-1 and EaNat-2 primer sets were specific for isolates of the natsudaidai pathotype. The Efaw-1 and Efaw-2 primer sets successfully detected E. fawcettii from lesions on diseased leaves and fruit from Korea and primer pairs Eaut-1, Eaut-2, Eaut-3, Eaut-4, and Eaut-5 detected E. australis from lesions on sweet orange fruit from Brazil.

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Palindromic sequence-targeted (PST) PCR: a rapid and efficient method for high-throughput gene characterization and genome walking

Scientific Reports ◽

10.1038/s41598-019-54168-0 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 6

Author(s):

Ruslan Kalendar ◽

Alexandr V. Shustov ◽

Mervi M. Seppänen ◽

Alan H. Schulman ◽

Frederick L. Stoddard

Keyword(s):

Thermal Cycling ◽

Annealing Temperature ◽

Phleum Pratense ◽

Genome Walking ◽

Palindromic Sequence ◽

Linear Amplification ◽

Gene Characterization ◽

Intron 1 ◽

Thermal Cycling Profile ◽

Palindromic Sequences

AbstractGenome walking (GW) refers to the capture and sequencing of unknown regions in a long DNA molecule that are adjacent to a region with a known sequence. A novel PCR-based method, palindromic sequence-targeted PCR (PST-PCR), was developed. PST-PCR is based on a distinctive design of walking primers and special thermal cycling conditions. The walking primers (PST primers) match palindromic sequences (PST sites) that are randomly distributed in natural DNA. The PST primers have palindromic sequences at their 3′-ends. Upstream of the palindromes there is a degenerate sequence (8–12 nucleotides long); defined adapters are present at the 5′-termini. The thermal cycling profile has a linear amplification phase and an exponential amplification phase differing in annealing temperature. Changing the annealing temperature to switch the amplification phases at a defined cycle controls the balance between sensitivity and specificity. In contrast to traditional genome walking methods, PST-PCR is rapid (two to three hours to produce GW fragments) as it uses only one or two PCR rounds. Using PST-PCR, previously unknown regions (the promoter and intron 1) of the VRN1 gene of Timothy-grass (Phleum pratense L.) were captured for sequencing. In our experience, PST-PCR had higher throughput and greater convenience in comparison to other GW methods.

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First report of Coleus blumei viroid 1 in commercial Coleus blumei in the United States

Plant Disease ◽

10.1094/pdis-05-21-0917-pdn ◽

2021 ◽

Author(s):

Gardenia Orellana ◽

Alexander V Karasev

Keyword(s):

United States ◽

Leaf Tissue ◽

The United States ◽

Universal Primers ◽

Rt Pcr ◽

Universal Primer ◽

Tissue Samples ◽

First Report ◽

Specific Pcr ◽

Coleus Blumei

Coleus scutellarioides (syn. Coleus blumei) is a widely grown evergreen ornamental plant valued for its highly decorative variegated leaves. Six viroids, named Coleus blumei viroid 1 to 6 (CbVd-1 to -6) have been identified in coleus plants in many countries of the world (Nie and Singh 2017), including Canada (Smith et al. 2018). However there have been no reports of Coleus blumei viroids occurring in the U.S.A. (Nie and Singh 2017). In April 2021, leaf tissue samples from 27 cultivars of C. blumei, one plant of each, were submitted to the University of Idaho laboratory from a commercial nursery located in Oregon to screen for the presence of viroids. The sampled plants were selected randomly and no symptoms were apparent in any of the samples. Total nucleic acids were extracted from each sample (Dellaporta et al. 1983) and used in reverse-transcription (RT)-PCR tests (Jiang et al. 2011) for the CbVd-1 and CbVd-5 with the universal primer pair CbVds-P1/P2, which amplifies the complete genome of all members in the genus Coleviroid (Jiang et al. 2011), and two additional primer pairs, CbVd1-F1/R1 and CbVd5-F1/R1, specific for CbVd-1 and CbVd-5, respectively (Smith et al. 2018). Five C. blumei plants (cvs Fire Mountain, Lovebird, Smokey Rose, Marrakesh, and Nutmeg) were positive for a coleviroid based on the observation of the single 250-nt band in the RT-PCR test with CbVds-P1/P2 primers. Two of these CbVd-1 positive plants (cvs Lovebird and Nutmeg) were also positive for CbVd-1 based on the presence of a single 150-nt band in the RT-PCR assay with CbVd1-F1/R1 primers. One plant (cv Jigsaw) was positive for CbVd-1, i.e. showing the 150-nt band in RT-PCR with CbVd1-F1/R1 primers, but did not show the ca. 250-bp band in RT-PCR with primers CbVds-P1/P2. None of the tested plants were positive for CbVd-5, either with the specific, or universal primers. All coleviroid- and CbVd-1-specific PCR products were sequenced directly using the Sanger methodology, and revealed whole genomes for five isolates of CbVd-1 from Oregon, U.S.A. The genomes of the five CbVd-1 isolates displayed 96.9-100% identity among each other and 96.0-100% identity to the CbVd-1 sequences available in GenBank. Because the sequences from cvs Lovebird, Marrakesh, and Nutmeg, were found 100% identical, one sequence was deposited in GenBank (MZ326145). Two other sequences, from cvs Fire Mountain and Smokey Rose, were deposited in the GenBank under accession numbers MZ326144 and MZ326146, respectively. To the best of our knowledge, this is the first report of CbVd-1 in the United States.

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454-Pyrosequencing Analysis of Bacterial Communities from Autotrophic Nitrogen Removal Bioreactors Utilizing Universal Primers: Effect of Annealing Temperature

BioMed Research International ◽

10.1155/2015/892013 ◽

2015 ◽

Vol 2015 ◽

pp. 1-12 ◽

Cited By ~ 11

Author(s):

Alejandro Gonzalez-Martinez ◽

Alejandro Rodriguez-Sanchez ◽

Belén Rodelas ◽

Ben A. Abbas ◽

Maria Victoria Martinez-Toledo ◽

...

Keyword(s):

Bacterial Diversity ◽

Nitrogen Removal ◽

Annealing Temperature ◽

16S Rrna Genes ◽

Rrna Genes ◽

Universal Primers ◽

Anammox Bacteria ◽

Universal Primer ◽

Optimum Annealing Temperature ◽

Autotrophic Nitrogen Removal

Identification of anaerobic ammonium oxidizing (anammox) bacteria by molecular tools aimed at the evaluation of bacterial diversity in autotrophic nitrogen removal systems is limited by the difficulty to design universal primers for theBacteriadomain able to amplify the anammox 16S rRNA genes. A metagenomic analysis (pyrosequencing) of total bacterial diversity including anammox population in five autotrophic nitrogen removal technologies, two bench-scale models (MBR and Low Temperature CANON) and three full-scale bioreactors (anammox, CANON, and DEMON), was successfully carried out by optimization of primer selection and PCR conditions (annealing temperature). The universal primer 530F was identified as the best candidate for total bacteria and anammox bacteria diversity coverage. Salt-adjusted optimum annealing temperature of primer 530F was calculated (47°C) and hence a range of annealing temperatures of 44–49°C was tested. Pyrosequencing data showed that annealing temperature of 45°C yielded the best results in terms of species richness and diversity for all bioreactors analyzed.

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Comparison of Two Suspension Arrays for Simultaneous Detection of Five Biothreat Bacterial in Powder Samples

Journal of Biomedicine and Biotechnology ◽

10.1155/2012/831052 ◽

2012 ◽

Vol 2012 ◽

pp. 1-8 ◽

Cited By ~ 9

Author(s):

Yu Yang ◽

Jing Wang ◽

Haiyan Wen ◽

Hengchuan Liu

Keyword(s):

Bacillus Anthracis ◽

Yersinia Pestis ◽

Simultaneous Detection ◽

Universal Primers ◽

Universal Primer ◽

Suspension Array ◽

Specific Pathogen ◽

Powder Samples ◽

High Conservation ◽

Species Specific

We have developed novel Bio-Plex assays for simultaneous detection ofBacillus anthracis, Yersinia pestis, Brucella spp., Francisella tularensis,andBurkholderia pseudomallei. Universal primers were used to amplify highly conserved region located within the 16S rRNA amplicon, followed by hybridized to pathogen-specific probes for identification of these five organisms. The other assay is based on multiplex PCR to simultaneously amplify five species-specific pathogen identification-targeted regions unique to individual pathogen. Both of the two arrays are validated to be flexible and sensitive for simultaneous detection of bioterrorism bacteria. However, universal primer PCR-based array could not identifyBacillus anthracis, Yersinia pestis,andBrucella spp.at the species level because of the high conservation of 16S rDNA of the same genus. The two suspension arrays can be utilized to detectBacillus anthracissterne spore andYersinia pestisEV76 from mimic “write powder” samples, they also proved that the suspension array system will be valuable tools for diagnosis of bacterial biothreat agents in environmental samples.

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A PCR Method That Can Be Further Developed into PCR-RFLP Assay for Eight Animal Species Identification

Journal of Analytical Methods in Chemistry ◽

10.1155/2018/5890140 ◽

2018 ◽

Vol 2018 ◽

pp. 1-6 ◽

Cited By ~ 9

Author(s):

Feng Guan ◽

Yu-Ting Jin ◽

Jin Zhao ◽

Ai-Chun Xu ◽

Yuan-Yuan Luo

Keyword(s):

Species Identification ◽

Animal Species ◽

Limit Of Detection ◽

Cost Effective ◽

Universal Primers ◽

Universal Primer ◽

Routine Identification ◽

Pcr Rflp ◽

Fast Processing ◽

Rflp Assay

There are many PCR-based methods for animal species identification; however, their detection numbers are limited or could not identify unknown species. We set out to solve this problem by developing a universal primer PCR assay for simultaneous identification of eight animal species, including goat, sheep, deer, buffalo, cattle, yak, pig, and camel. In this assay, the variable lengths of mitochondrial DNA were amplified using a pair of universal primers. PCR amplifications yielded 760 bp, 737 bp, 537 bp, 486 bp, 481 bp, 464 bp, 429 bp, and 359 bp length fragments for goat, sheep, deer, buffalo, cattle, yak, pig, and camel, respectively. This primer pair had no cross-reaction with other common domestic animals and fish. The limit of detection varied from 0.01 to 0.05 ng of genomic DNA for eight animal species in a 20 µl PCR mixture. Each PCR product could be further digested into fragments with variable sizes and qualitative analysis by SspI restriction enzyme. This developed PCR-RFLP assay was sufficient to distinguish all targeted species. Compared with the previous published related methods, this approach is simple, with high throughput, fast processing rates, and more cost-effective for routine identification of meat in foodstuffs.

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666 PB 110 RANDOM AMPLIFIED POLYMORPHIC DNA MARKERS ARE INADEQUATE FOR FINGERPRINTING GRAPE ROOTSTOCKS

HortScience ◽

10.21273/hortsci.29.5.528c ◽

1994 ◽

Vol 29 (5) ◽

pp. 528c-528

Author(s):

Alan T. Bakalinsky ◽

Hong Xu ◽

Diane J. Wilson ◽

S. Arulsekar

Keyword(s):

Dna Markers ◽

Rapd Markers ◽

Restriction Site ◽

Random Amplified Polymorphic Dna ◽

Rapd Marker ◽

Base Length ◽

Specific Primer ◽

Annealing Temperatures ◽

Individual Reaction ◽

Length Variant

A total of eight random amplified polymorphic DNA (RAPD) markers were generated in a screen of 77 primers of 10-base length and were detected reproducibly among nine different grape (Vitis) rootstocks. Occasional failed amplifications could not be explained rationally nor easily corrected by systematic replacement of individual reaction components. In an effort to improve their reliability, the RAPD markers were cloned, their termini sequenced, and new sequence-specific primer pairs were synthesized based on addition of 10 to 14 bases to the 3' termini of the original 10-mers. Six pairs of the new primers were evaluated at their optimal and higher-than optimal annealing temperatures. One primer pair amplified a product the same size as the original RAPD marker in all rootstocks, resulting in loss of polymorphism. Post-amplification digestion with 7 different restriction endonucleases failed to reveal restriction site differences. Three primer pairs amplified an unexpected length variant in some accessions. Two other pairs of primers amplified a number of unexpected bands. Better approaches for exploiting the sequence differences that account for the RAPD phenomenon will be discussed.

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Using Random Amplified Polymorphic DNA (RAPD) analysis to investigation of genetic diversity, and relationships among a set of clinical Aspergillus fumigatus isolates

Journal of Biotechnology Research Center ◽

10.24126/jobrc.2014.8.1.304 ◽

2014 ◽

Vol 8 (1) ◽

pp. 46-54

Author(s):

Batool Omran Theeb ◽

Abdulkareem Jasim Hashim ◽

Akeel Hussain Ali Al-Assi

Keyword(s):

Genetic Diversity ◽

Aspergillus Fumigatus ◽

Genetic Relatedness ◽

Random Amplified Polymorphic Dna ◽

Geographic Origin ◽

Close Relation ◽

Genetic Distances ◽

Universal Primers ◽

Characteristic Banding ◽

High Level

This study is an attempt to determine the genetic diversity and relationships among fourteen local isolate isolated from patients with Aspergillosis (Aspergillus fumigatus) by using the Random Amplified Polymorphic DNA (RAPD) technique. Twelve universal primers used in this study produced 94 bands across fourteen isolates. Of these bands, 67 bands or 71.2% were polymorphic. The size of the amplified bands ranged between 100-2000 bp. The genetic polymorphism value of each primer was determined and ranged between 33-100%. In terms of unique banding patterns, determine the finger print for six isolates the most characteristic banding pattern was for the (AFU1, AFU2, AFU3, AFU4, AFU8 and AFU14) with primer (OP F-16 , OP I-06, OP F-16, OP X-01, OP X-01and OP A-06). Genetic distances ranged from 0.12419 to 0.64404 among A. fumigatus isolates. Cluster analyses were performed to construct a dendrogram among studied A. fumigatus isolates. The cluster analysis places most of the A.fumigatus isolates isolated from patient come from yhe same area into a close relation (subcluster) showing a high level of genetic relatedness and were distinct from isolates from another area (the other subcluster). Interestingly, a number of isolates originating from the same sources did form well defined groups, indicating association between the RAPD patterns and the geographic origin of the isolates. The information generated from this study can be used in the future for controlling of Aspergillosis programs.

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Artificial DNA Templates for the Correction of PCR Bias in Next Generation Sequencing Based MRD Analysis for Childhood Acute Lymphoblastic Leukaemia: The Influence of Secondary DNA Structure

Blood ◽

10.1182/blood.v128.22.4078.4078 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4078-4078

Author(s):

John Moppett ◽

Jerry Hancock ◽

Marc Duez ◽

Jack Bartram ◽

Gary Wright ◽

...

Keyword(s):

Secondary Structure ◽

Universal Primers ◽

Amplification Efficiency ◽

Minimal Risk ◽

Universal Primer ◽

Dna Templates ◽

Pcr Bias ◽

Artificial Dna

Abstract PCR bias is a potential confounder for PCR-based NGS-MRD quantitation. A method was published using artificial DNA constructs (gBlocks, IDT Technologies, Coralville, IO) to assess PCR primer bias and correct for it(1). We sought to confirm those findings by assessing PCR bias of the Biomed-2 TRG primer set. 36 synthetic DNA templates of 495bp length gBlocks combining each TRG V (V 2,3,4,5,7,8,9,10,11) & J gene (JG1-01, JG1-02, JP1, JP2) amplified by the Biomed-2 primer set were synthesised containing external MiSeq flow cell adaptor sequence (universal primer (UP)) binding sites, VG and JG sequence and junctional regions. Barcodes inserted internal to the universal primer sites and centrally enabled accurate template identification (fig 1). Individual gBlocks supplied at a nominal concentration of 10ng/uL (30nM) were quantitated by TapeStation (Agilent, Santa Clara, CA) and KAPA (Kapa Biosystems, Wilmington, MA) methodologies and pooled (unadjusted). gBlockswithin the pool were further quantitated by both 6 and 8 cycles of PCR with universal MiSeq adaptor primers. Next the gBlock pool was amplified in triplicate in a 2-stage PCR process: 1) Using Biomed-2 TRG primers that contained partial MiSeq adaptor sequences. The resulting products were purified with Agencourt AMPure XP beads (Beckman Coulter, Jersey City, NJ). 2) These amplicons were further amplified by primers containing indices and full MiSeq adaptor sequences. After purification, amplicons were Qubit (InvitroGen, Carlsbad, CA) and TapeStation analysed for normalisation and to construct the sequencing library, which was KAPA quantitated to ensure ideal cluster density. Bi-directional sequencing was performed using an Illumina MiSeq 500 cycle cartridge and Nano Reagent Kit (Illumina, San Diego, CA). Bioinformatics analysis was performed using the Vidjil platform (Bonsai team, CRIStAL, Lille, France). TapeStation quantitation showed a mean concentration of 3.3ng/uL (10nM) (range 1.1-6.3ug/uL). KAPA quantitation by contrast showed low quantities of all gBlocks (2nM, 0.67ug/uL) with the exception of all VG9 and most VG10 constructs (16nM, 5ug/uL)(fig 2a). Comparison of 6 vs 8 cycles of amplification using universal primers showed good correlation (R2=0.991) confirming that PCR cycle length does not affect the UP amplification (fig 2b), but there was an 8.5-fold variation in gBlock quantitation by UP PCR. There was no correlation between NGS quantitation and with either TapeStation or KAPA quantitation. Family specific V & J gene amplification efficiency for the Biomed-2 primers is shown in fig 3. Amplification efficiencies of V gene primers were <2-fold different with the exception of VY9 and VY10 which were 5-fold more efficient. Amplification efficiency for J gene primers showed <2-fold differences. This study has shown that there are clear PCR efficiency differences between the V primers in the Biomed-2 primer set, with VG9 and 10 being 5x more efficient. This could be adjusted for by reducing the concentrations of the VG9/10 primers. However, the variable results of the 3 methods used to quantitate the artificial templates raises significant questions about the rationale. TapeStation quantitation is not PCR based and produced broadly equivalent (but lower than anticipated) gBlock concentrations for the individual constructs. PCR based KAPA showed very low concentrations in all but the VG9/VG10 templates. It is notable that this exactly parallels the results for the locus specific PCR. The most plausible explanation is that unexpected secondary structure reduces the amplifiability of the templates in PCR reactions. The near 10-fold variation in template concentration as assessed by UP NGS-PCR is concerning, suggesting significant secondary structure effects, though not dissimilar to the 5-fold difference seen in the published method(1). It is impossible to know if the postulated secondary structure effect that we see is replicated in vivo. In that regard it is reassuring that published results of NGS-MRD appear to correlate well with RQ-PCR results. In conclusion, secondary structure issues potentially affect the in vitro method for eliminating PCR bias. Within the EuroClonality consortium, studies at other loci are underway to confirm these preliminary findings, and to design primer sets with minimal risk of PCR bias. 1. Carlson CS, Emerson RO, Sherwood AM et al. Nat Commun. 2013;4:2680. Disclosures Moppett: Jazz Pharmaceuticals: Honoraria.

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