Multiplex primer prediction software for divergent targets

We describe a Multiplex Primer Prediction (MPP) algorithm to build multiplex compatible primer sets to amplify all members of large, diverse and unalignable sets of target sequences. The MPP algorithm is scalable to larger target sets than other available software, and it does not require a multiple sequence alignment. We applied it to questions in viral detection, and demonstrated that there are no universally conserved priming sequences among viruses and that it could require an unfeasibly large number of primers (∼3700 18-mers or ∼2000 10-mers) to generate amplicons from all sequenced viruses. We then designed primer sets separately for each viral family, and for several diverse species such as foot-and-mouth disease virus (FMDV), hemagglutinin (HA) and neuraminidase (NA) segments of influenza A virus, Norwalk virus, and HIV-1. We empirically demonstrated the application of the software with a multiplex set of 16 short (10 nt) primers designed to amplify the Poxviridae family to produce a specific amplicon from vaccinia virus.

[1]  Elizabeth A. Kellogg,et al.  Primaclade - a flexible tool to find conserved PCR primers across multiple species , 2005, Bioinform..

[2]  A. Vaheri,et al.  Detection of human orthopoxvirus infections and differentiation of smallpox virus with real‐time PCR , 2009, Journal of medical virology.

[3]  R. Edwards,et al.  Viral metagenomics , 2005, Nature Reviews Microbiology.

[4]  D. Cavanagh.  SARS- and Other Coronaviruses , 2008, Methods in Molecular Biology.

[5]  Robert Giegerich,et al.  BMC Bioinformatics BioMed Central Methodology article Efficient computation of absent words in genomic sequences , 2008 .

[6]  M. Magnani,et al.  Fast and sensitive quantitative detection of HIV DNA in whole blood leucocytes by SYBR green I real-time PCR assay. , 2007, Molecular and cellular probes.

[7]  Simon N. Jarman,et al.  Amplicon: software for designing PCR primers on aligned DNA sequences , 2004, Bioinform..

[8]  G. Palacios,et al.  Diagnostic System for Rapid and Sensitive Differential Detection of Pathogens , 2005, Emerging infectious diseases.

[9]  S. Shchelkunov,et al.  Real-Time PCR System for Detection of Orthopoxviruses and Simultaneous Identification of Smallpox Virus , 2004, Journal of Clinical Microbiology.

[10]  David A Stenger,et al.  Broad-spectrum respiratory tract pathogen identification using resequencing DNA microarrays. , 2006, Genome research.

[11]  K. Livak,et al.  DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. , 1990, Nucleic acids research.

[12]  P. Krause,et al.  Universal virus detection by degenerate-oligonucleotide primed polymerase chain reaction of purified viral nucleic acids , 2008, Journal of Virological Methods.

[13]  Benjamin J Hindson,et al.  On-chip, real-time, single-copy polymerase chain reaction in picoliter droplets. , 2007, Analytical chemistry.

[14]  Kevin L. Russell,et al.  Using a Resequencing Microarray as a Multiple Respiratory Pathogen Detection Assay , 2006, Journal of Clinical Microbiology.

[15]  B. Berkhout,et al.  Human Parechovirus Type 1, 3, 4, 5, and 6 Detection in Picornavirus Cultures , 2007, Journal of Clinical Microbiology.

[16]  G. Palacios,et al.  Rapid sequence-based diagnosis of viral infection. , 2008, Antiviral research.

[17]  Simon Kasif,et al.  Computational tradeoffs in multiplex PCR assay design for SNP genotyping , 2005, BMC Genomics.

[18]  Sean R. Eddy,et al.  Profile hidden Markov models , 1998, Bioinform..

[19]  G. Palacios,et al.  Multiplex MassTag-PCR for respiratory pathogens in pediatric nasopharyngeal washes negative by conventional diagnostic testing shows a high prevalence of viruses belonging to a newly recognized rhinovirus clade , 2008, Journal of Clinical Virology.

[20]  Cheng-Yan Kao,et al.  Integrated minimum-set primers and unique probe design algorithms for differential detection on symptom-related pathogens , 2005, Bioinform..

[21]  William R. Pearson,et al.  A New Approach to Primer Selection in Polymerase Chain Reaction Experiments , 1995, ISMB.

[22]  David L. Hirschberg,et al.  Detection of Respiratory Viruses and Subtype Identification of Influenza A Viruses by GreeneChipResp Oligonucleotide Microarray , 2007, Journal of Clinical Microbiology.

[23]  Robert C. Edgar,et al.  MUSCLE: multiple sequence alignment with high accuracy and high throughput. , 2004, Nucleic acids research.

[24]  Ron Shamir,et al.  Degenerate Primer Design , 2007 .

[25]  J. Kawai,et al.  Direct Metagenomic Detection of Viral Pathogens in Nasal and Fecal Specimens Using an Unbiased High-Throughput Sequencing Approach , 2009, PloS one.

[26]  Timothy Rose,et al.  CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primer) PCR primer design , 2003, Nucleic Acids Res..

[27]  G. Palacios,et al.  MassTag Polymerase-Chain-Reaction Detection of Respiratory Pathogens, Including a New Rhinovirus Genotype, That Caused Influenza-Like Illness in New York State during 2004–2005 , 2006, The Journal of infectious diseases.

[28]  James B Thissen,et al.  Multiplexed molecular assay for rapid exclusion of foot-and-mouth disease. , 2008, Journal of virological methods.

[29]  Rangarajan Sampath,et al.  Rapid Identification of Emerging Infectious Agents Using PCR and Electrospray Ionization Mass Spectrometry , 2007, Annals of the New York Academy of Sciences.

[30]  S. Gardner,et al.  Annals of Clinical Microbiology and Antimicrobials Open Access Predicting the Sensitivity and Specificity of Published Real-time Pcr Assays , 2008 .

[31]  Yang Liu,et al.  Panmicrobial Oligonucleotide Array for Diagnosis of Infectious Diseases , 2007, Emerging infectious diseases.

[32]  W. Ian Lipkin,et al.  Greene SCPrimer: a rapid comprehensive tool for designing degenerate primers from multiple sequence alignments , 2006, Nucleic acids research.

[33]  E. Delwart,et al.  Metagenomic Analyses of Viruses in Stool Samples from Children with Acute Flaccid Paralysis , 2009, Journal of Virology.

[34]  Michael Zuker,et al.  DINAMelt web server for nucleic acid melting prediction , 2005, Nucleic Acids Res..

[35]  Rodrigo Lopez,et al.  Multiple sequence alignment with the Clustal series of programs , 2003, Nucleic Acids Res..

[36]  G. Pesole,et al.  GeneUp: a program to select short PCR primer pairs that occur in multiple members of sequence lists. , 1998, BioTechniques.

[37]  Ron Shamir,et al.  The Degenerate Primer Design Problem , 2002, ISMB.

[38]  J. Derisi,et al.  Pan-Viral Screening of Respiratory Tract Infections in Adults With and Without Asthma Reveals Unexpected Human Coronavirus and Human Rhinovirus Diversity , 2007, The Journal of infectious diseases.

[39]  D. Ecker,et al.  The Ibis T5000 Universal Biosensor: An Automated Platform for Pathogen Identification and Strain Typing , 2006 .

[40]  J. Derisi,et al.  Microarray-based detection and genotyping of viral pathogens , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[41]  Simon Kasif,et al.  MuPlex: multi-objective multiplex PCR assay design , 2005, Nucleic Acids Res..

[42]  G. Caetano-Anollés,et al.  DNA Amplification Fingerprinting Using Very Short Arbitrary Oligonucleotide Primers , 1991, Bio/Technology.

[43]  J. Fox.  Nucleic acid amplification tests for detection of respiratory viruses , 2006, Journal of Clinical Virology.

[44]  Ludwig Eichinger,et al.  Large scale multiplex PCR improves pathogen detection by DNA microarrays , 2009, BMC Microbiology.