2012 Annual Report
1a.Objectives (from AD-416):
To demonstrate the ability of an oligonucleotide microarray to detect and differentiate plant viruses from random amplification of plant total nucleic acid extracts, and to provide education, through training, of both undergraduate interns and a graduate student, and the incorporation of microarray techniques into curricula.
1b.Approach (from AD-416):
ARS will acquire the lists of viral taxa to be represented on the viral detection microarray, and virus-infected samples from which to amplify nucleic acids to validate the microarray. This information and material will be utilized by both ARS and the Cooperator to jointly develop and validate the microarray for detection of target viruses, and to make validation results available to collaborators via a web server. The Cooperator will perform analysis of viral sequences to identify suitable sequences for the development of oligonucleotides, and participate in analysis of microarray hybridization results to determine with a high degree of confidence which viruses were present in validation samples. The Cooperator will also integrate microarray techniques into curricula, and educate students on the theory and application of microarrays.
The Universal Plant Virus Microarray (UPVM) oligo sequences were utilized as E-probes to identify virus sequences in high-throughput sequencing data. Samples of putative virus-infected plants were subjected to high-throughput sequencing, and the resulting sequence contigs were analyzed separately based solely on the contig sequence, and by using BLASTn to screen the contigs against the UPVM oligo sequences. A total of 251 plants were identified as being virus infected based solely on the contig sequence, an additional 294 plants by both contig sequence and BLASTn with the UPVM oligo sequences, and no virus was detected by either method in a total of 780 plants. Notably no “UPVM oligo-only” false positives were registered, yielding a sensitivity of 53.9%, a specificity of detection of 100%, and an accuracy of identification of 81.1%. Possible reasons for failure of BLASTn using the UPVM oligos to identify all samples in which virus was detected by sequence analysis include: a) poor coverage of the viral genome in the sequence data due to low titer of the virus; and b) biased synthesis of viral cDNA from nucleic acid extracts of infected plants due to preferential priming or nucleic acid secondary structure. It was observed that the ‘tag’ sequence of ‘tagged’ random primers contributed to primer binding and cDNA initiation even though the primers included 12 random nucleotides downstream of the conserved ‘tag’ sequence. Further work is utilizing the UPVM probes to search the database of singleton contigs from multiple plants, in an iterative assembly and query of the singleton database, to attempt to build a consensus contig of a virus (incompletely represented in data from any single plant) from multiple plant sources.
In related work on a different microarray system, it was necessary to fragment amplified viral sequences for efficient hybridization, whereas the UPVM protocols utilized non-amplified, non-fragmented cDNA. Inferences drawn from these experiments are:.
1)That the potential sensitivity of microarray hybridization is comparable to moderate density high-throughput sequencing;.
2)Long amplified targets bind poorly to an array of tailed oligo probes;.
3)95% of existing viruses did not contribute directly to the design of the UPVM probes (since only 5% of putative viruses identified in a sequence-based survey corresponded to viral species known to science); and.
4)tagged primer methods of cDNA production or amplification introduce biases that can interfere with detection. These results were presented at the UPVM Workshop and BARD Workshop ‘Microarrays and Next-Generation Sequencing for Detection and Identification of Plant Viruses’ held at Beltsville in November 2011. This information will be of most immediate application to the UPVM collaborators, but will also be of value to regulatory agencies, plant diagnostic clinics, germplasm repositories, and producers operating plant certification schemes.