Progress 12/13/18 to 10/31/20
Outputs
Target Audience:Fish health professionals Changes/Problems:Covid 19 associated shutdown and quarantine of personnel delayed research. What opportunities for training and professional development has the project provided?The laboratory manager received training on the use of the Nanopore sequencer and data analysis. She is also the primary laboratory associate that performs viral and molecular diagnostic assays for the MSU/CVM Aquatic Diagnostic Laboratory. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objectives: The primary goal of this proposal is to evaluate the use of Nanopore sequencing in the fish diagnostic laboratory. Objective 1: Optimizing diagnostic sample preparation and analysis. We plan to evaluate tissue extract and cell culture supernatant paired samples from 2 known virus infected samples. Objective 2: Application of the optimized method to real diagnostic cases. At least 6 different fish diagnostic cases will be evaluated. The nanopore starter kit was purchased with the rapid barcoding sequencing kit. Lorelei Ford, research associate, obtained training on using the Nanopore device and data analysis. The nucleic acid extraction, processing and sequencing was modeled on the method of McCabe et al 2018. This was used with DNA extracted from Ictalurid herpesvirus 1 (IcHV1) infected channel catfish ovary cells (CCO), Largemouth bass virus (LMBV) infected fathead minnow cells (FHM) and shrimp tissue infected with infectious hypodermal and hematopoietic necrosis virus (IHHNV- Decapod penstyldensovirus 1). The purification process consisted of centrifuging the tissue or cell culture lysate at 1000xG for 15 minutes to pellet out cellular debris. Then the supernatant was treated with RNaseA and Turbo DNase to remove non-viral DNA. Following this, nucleic acids were extracted using QIAamp Ultrasense VIRAL RNA/DNA Purification kit. The nucleic acids were quantified using the Nanodrop and the DNA was prepared for sequencing using the tranposase based- nanopore rapid barcoding kit. In the first and second tries, large amount of sequence (over 1000 fold sequence coverage was obtained from IcHV1 but no sequences were detected from the LMBV and IHHNV. The second run gave well-over 1000 IcHV1 matches in less than 7 minutes. The multiplex procedures did not give a distribution of sequences. Also, we were concerned that the IHHNV sample was not working because this virus is a Parvovirus. Its genome is single stranded and small 4,000 bp. Therefore, we evaluated using a random hexamer primed double stranded cDNA synthesis kit before sequencing (Thermo Scientific Maxima H Minus Double-Stranded cDNA Synthesis Kit). A third run was done using just LMBV and IHHNV used directly of via the cDNA reaction. This run gave over 1000 fold coverage of the LMBV genome but no IHHNV sequence. We are now evaluating the potential that the Nanopore sequencing kit is optimized for long DNA sequences (IHHNV has a very small genome). To optimize sequencing of small ssDNA genomes and ssRNA genomes we used canine parvovirus (CPV) from diagnostic samples sent to us from Jackson after culture on MDCK cells and Bovine respiratory syncytial virus (BRSV). On the first try using the rapid barcoding kit after dscDNA synthesis. We got 39 BRSV and 15 CPV sequences. This was repeated using the PCR barcoding kit. This yielded 85 BRSV and 4 CPV sequences. Significant results achieved- The study demonstrated that the nanopore sequencing can give good quality sequence data on large DNA viruses very quickly with relatively little processing. However small ssRNA and ssDNA viruses yielded much lower numbers of sequences and the sequencing analysis parameters needed to be lowered to recognized smaller fragments. Within each sequencing run there were large numbers of host DNA sequences and in early trials we found considerable numbers of human, bacteria and fungus sequences. To reduce background sequences, we changed our protocol so that most steps in handling nucleic acid are done in a BSC (after UV treatment and alcohol wiping) with a lab coat and gloves. For any benchwork all surfaces are wiped down with 70% isopropanol and then sprayed with 5% hydrogen peroxide. Furthermore, a considerable amount of read-through occurred from previous runs on the nanopore device so all new runs must use new barcodes and only the expected barcoded DNA evaluated. In our trials we compared our sequences reference genomes of the pathogen and the host. We also evaluate the use of What's in My Pot (WIMP) software while basecalling but this program does not BLAST shorter sequences and not all pathogen sequences from GenBank are in the database. For example, LMBV was not in the WIMP database and sequences returned similarity to another Ranavirus. We found that for large DNA viruses the protocol was relatively easy and provided good data and was amenable to routine use in a diagnostic setting, however for yield for RNA viruses and ssDNA viruses was too low to be practical or trusted, the future we will evaluate virus concentration/purification steps to enhance this yield. This must be done before the procedure can be established that would allow detection and sequence characterization of various virus types with no previous knowledge needed about the virus. If successful, the nanopore system could be used as a comprehensive diagnostic tool for viral disease diagnostics. Reference: Mccabe, M., Cormican, P., Johnston, D. & Earley, B. (2018) Simultaneous detection of DNA and RNA virus species involved in bovine respiratory disease by PCR-free rapid tagmentation-based library preparation and MinION nanopore sequencing. bioRxiv, 269936..
Publications
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Progress 12/13/18 to 09/30/19
Outputs
Target Audience:The target audience on this project is fish health professionals. Changes/Problems:There are large differences in the efficiency of sequencing different viruses and this will affect the reliability of detecting viruses in diagnostic samples. We are consulting with nanopore and others that use nanopore sequencing to optimize sequencing efficiency for both large and small genomes. What opportunities for training and professional development has the project provided?The laboratory manager received training on the use of the Nanopore sequencer and data analysis. She is also the primary laboratory associate that performs viral and molecular diagnostic assays for the MSU/CVM Aquatic Diagnostic Laboratory. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?Repeat assays using single stranded RNA viruses, and double stranded RNA viruses and evaluate options on the sequencing kit to sequence small DNA products.
Impacts
What was accomplished under these goals?
Objectives: The primary goal of this proposal is to evaluate the use of Nanopore sequencing in the fish diagnostic laboratory. Objective 1: Optimizing diagnostic sample preparation and analysis. We plan to evaluate tissue extract and cell culture supernatant paired samples from 2 known virus infected samples. Objective 2: Application of the optimized method to real diagnostic cases. At least 6 different fish diagnostic cases will be evaluated. The nanopore starter kit was purchased with the rapid barcoding sequencing kit. Lorelei Ford, research associate, obtained training on using the Nanopore device and data analysis. This was used with DNA extracted from Ictalurid herpesvirus 1 (IcHV1) infected channel catfish ovary cells (CCO), Largemouth bass virus (LMBV) infected fathead minnow cells (FHM) and shrimp tissue infected with infectious hypodermal and hematopoietic necrosis virus (IHHNV- Decapod penstyldensovirus 1). In the first and second tries, large amount of sequence (over 1000 fold sequence coverage was obtained from IcHV1 but no sequences were detected from the LMBV and IHHNV. The second run gave well-over 1000 IcHV1 matches in less than 7 minutes. The multiplex procedures did not give a distribution of sequences. Also, we were concerned that the IHHNV sample was not working because this virus is a Parvovirus. Its genome is single stranded and small 4,000 bp. Therefore, we evaluated using a double stranded cDNA synthesis kit before sequencing. A third run was done using just LMBV and IHHNV used directly of via the cDNA reaction. This run gave over 1000 fold coverage of the LMBV genome but no IHHNV sequence. We are now evaluating the potential that the Nanopore sequencing kit is optimized for long DNA sequences (IHHNV has a very small genome). Significant results achieved- The study demonstrated that the nanopore sequencing can give good quality sequence data very quickly with relatively little processing. Thus, it is amenable to routine use in a diagnostic setting. The next step of this study is to identify a procedure that will allow detection and sequence characterization of various virus types with no previous knowledge needed about the virus, so that it can be used as a comprehensive diagnostic tool for viral disease diagnostics.
Publications
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