Progress 11/29/18 to 09/30/20
Outputs
Target Audience:The audience are diagnostic technicians who are interested in RNA virus sequencing on MinION and iSeq100, poultry veterinarian and poultry industry who are interested in determining the serotypes of infectious bronchitis virus and genotype of avian reoviruses. Changes/Problems:At the beginning, we plan to use MinION to sequence all isolates, after we purchased iSeq100, I am interested in comparing the sequencing using iSeq100 and MinION. Therefore, sequencing was performed on both iSeq100 and MinION. Based on sequencing results, iSeq100 generate more reliable data. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?Sequencing of IBV were presented on AAVLD annual meeting at 2019, however, failed to get whole genome sequencing of reoviruses, so we did not present this part in either meeting or conference. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
We did try MinION and Illumina iSeq100 on sequencing infectious bronchitis virus and avian reoviruses. Based on our sequencing results, both MinION and iSeq100 generate good results for IBV, however, the sequencing results of reovirus are not as good as we expected. More than 10 reoviruses were sequenced using either iSeq100 or MinION, but the results are not as good as expected. We are able to get partial sequences of different segments, but we did not get the full genome sequences. Avian reovirus is segmented RNA viruses, therefore it gets difficult to sequence. Also the reovirus was isolated from chicken liver or kidney cells, there are lots of host genome sequences exists in the results. We did try two different ways to process the nucleic acids before library prep, but we still unable to get whole genome sequences. This might be related with the low output of iSeq100. We plan to try some commercial ribosomal depletion kits to preprocess RNA before sequencing. Also I tried sequence small numbers of isolates per run for reoviruses (4 isolates per run), I didn't get enough data to get full genome sequences of reovirus. More than 20 IBV isolates were sequenced successfully using either iSeq100 or MinION. This might related with the low host cells, because we use chicken embryonated eggs to isolate IBV and collect allantoic fluid to extract RNA. For the virus isolated from cell lines or tissues, large amount of data might be needed to generate the whole genome sequences. A change in action: Based on the project experience, we might start offering whole genome sequencing service for poultry industry or other researchers in the future.
Publications
|
Progress 11/29/18 to 09/30/19
Outputs
Target Audience:The audience are diagnostic technicians who are interested in RNA virus sequencing on MinION, poultry veterinarian and poultry industry who are interested in determining the serotypes of infectious bronchitis virus and genotype of avian reoviruses. ? Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Training activities: Nanopore introduction workshop and Data analysis overview April 18 to 19, 2019 in New York City. Professional development activities: present this study at AAVLD meeting. How have the results been disseminated to communities of interest?Presentation at AAVLD annual meeting, and discussed with poultry veterinarians in the same institutions. What do you plan to do during the next reporting period to accomplish the goals?For this study, we plan to sequence 20 IBV isolates and 20 reovirus isolates. Now we did sequence 10 IBV isolates and 1 reovirus isolates, and we are planning to sequence at least 10 more IBV isolates. So far we did not have a good results for reovirus, and we are planning to change the preprocessing procedure for sequencing of reovirus. And we plan to sequence 20 isolates reovirus. And standard protocol will be established to sequencing RNA viruses using MinION. ?
Impacts
What was accomplished under these goals?
1) The whole genome sequences of 7 IBV isolates and 3 partial sequences of IBV isolates were completed. Partial sequence of one reovirus isolate was received from sequencing. 2) Successfully sequencing of IBV on MinION sequencing machine. 3) As we expected, whole genome sequencing can be used to sequence and determine the serotype of IBVs. Especially for those isolates we are unable to amplify using primers we used in conventional PCR. However, reovirus as a segmented, dsRNA virus is difficult to sequence and get good quality of data. We are thinking to try to use ribosomal depletion to avoid the effect of cell lines used to isolate reovirus. 4) Whole genome sequence of one IBV isolate were completed, which is unable to amplify and sequence using either set of primes from our current conventional PCR. A change in knowledge: A change in action: Based on the project experience, we might start offering whole genome sequencing service for poultry industry or other researchers in the future.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2019
Citation:
Yan L, Mackey R, Zhang C, Banda A, Baughman S, Pace L. Application and Comparison of iSeq100 and MinION Sequencing for Identification of Avian Infectious Bronchitis Virus (IBV). American Association of Veterinary Laboratory Diagnosticians annual meeting. Oct 23-28, 2019. Providence RI.
|
|