B--IHNV GENOTYPING PROGRAM UPGRADE

The purpose of this amendment is to answer vendor questions. The period for questions is closed, and no further questions will be accepted. The due date for quotes remains the same, September 8, 2026, at 10:00am PDT.

1. How many data entries are currently in the database? approximately 3000 2. How many samples need to be added to the database from recent years? How many samples per year are being added Approximately 400. The number per year is not a set number but varies depending on field events and how many virus isolates our fish health partners to send in for genotyping.

3. Shall the previously used database stay in place? Or should the deliverables include a plan for long term database plan which may include development of a new structure/system? At this point we are aiming to update the current in-house excel database that has been in place for many years. That database will continue to be our main source of curated information. Future plans for any new system are desirable but beyond the financial scope of this contract.

4. Where (what lab) is the genotyping data being produced currently? The majority of genotyping is done at our lab, the USGS Western Fisheries Research Center, where the genotyping program originated and has been hosted since the 1990s. We also occasionally get isolate metadata and genotyped sequences from partners at the Northwest Indian Fish Commission and Oregon Department fo Fish and Wildlife.

5. What technology is being used for that? Microarray? Sanger sequencing? Next generation sequencing? Other? RNA is extracted from each virus isolate and a fragment containing the midG sequence is generated by PCR. The genotype consensus sequences are generated by Sanger or NGS either in-house or by sending the PCR fragement to a commercial sequencing service. Metadata is tracked using the in-house database that also contains the sequence generated. and the designated genotype number.

6. What is the size of each genotype that will be added or produced? Is it the full IHNV sequence or just select regions? IHNV genotypes have been established in North America using a 303 nt mid-G region that is a variable region in the virus glycoprotein (G) gene. This short region has proven robust in comparison with phylogenies based on complete G genes (1610 nt) or full genomes ( ~ 11,132 nt). It is the only sequence that facilitates phylogenetic analyses with archive of genotype data for over 3000 virus isolates that represent IHNV in all of North America.

7. What database engine does the current database use (e.g., PostgreSQL, mySQL, etc.)? The current database is a large excel file containing all metadata associated with isolates and the genotyped sequence that is associated with each isolate.

8. How many WFRC staff need to be trained annually to conduct the IHNV typing? Once staff are trained they do not need to be trained annually but as needed when any changes in the system need to be implemented. Optimally there should be two staff members who are trained to conduct the IHNV genotyping. ********************************************