Samples Received – Pinto Abalone DNased RNA from UC-Irvine

Received DNased pinto abalone RNA from Alyssa Braciszewski at UC-Irvine. These are subset of the samples I sent her back in February.

Here’s the samples list provided by Alyssa (Google Sheet): shipment to UW of RNA samples.xlsx

The samples need to be confirmed to be free if residual RLO gDNA via qPCR. If they are clean, then will proceed to making cDNA, using provided reagents.

Reagents were stored in door of -20C in FSH 240.

Samples were stored in the provided box in the “new” -80C in FSH 235.

Sanger Sequencing – pCR2.1/OsHV-1 ORF117 Sequencing Data

Received the Sanger sequencing data back from Genewiz for the samples I submitted last week.

AB1 files were downloaded as a zip file and stored in the Friedman Lab server: backupordie/lab/sequencing_data/Sanger/30-19717124_ab1.zip

Files were analyzed using Geneious 10.2.3.

Geneious analysis was exported (compatible with version 6.0 and up) and saved to the Friedman Lab server:

backupordie/lab/sam/Sequencing_Analysis/Sanger/20170821_oshv_orf117_sanger.geneious

Results:

After vector ID and trimming, all sequences from both colonies were aligned, resulting in an 867bp contig. The size of this contig jives perfectly with the bright PCR band at ~1000bp I saw when screening the two colonies (the ~1000bp includes 300bp of vector sequence from using the M13 primers).

 

The alignment above shows that there were no gaps in the sequencing between the two sequencing primers (M13 forward and M13 reverse). I point this out because the insert in this plasmid was supposed to be the full-length OsHV-1 ORF117 (which is ~1300bp), as described in: Detection of undescribed ostreid herpesvirus 1 (OsHV-1) specimens from Pacific oyster, Crassostrea gigas. Martenot et al. 2015. As the sequencing shows, that is not what is cloned in this vector.

To determine what was actually cloned in this vector, I performed a BLASTx against the nr database, using the consensus sequence generated from the alignment above:

 

BLASTx generated a total of six matches, five of which match OsHV-1 ORF117 (the hypothetical and RING finger proteins listed above actually have alternate accession numbers that all point to ORF117). However, notice in the one alignment example provided at the bottom of the above image, the Query (i.e. our consensus sequence) only starts aligning at nucleotide 109 and matches up with the NCBI OsHV-1 ORF117 beginning at amino acid 158.

The results clearly show that the insert in this vector is OsHV-1 ORF117, but it is not the entire thing. To confirm this, I aligned the consensus sequence to the OsHV-1 genome (GenBank: AY509253.2) using Geneious:

 

In the image above, I have zoomed into the region in which our sequencing consensus aligned within the OsHV-1 genome. In order to see in more detail, please click on the image above. There are two noticeable things in this alignment:

  1. The insert we sequenced doesn’t span the entire ORF117 coding sequence (the yellow annotation in the image above).

  2. There’s a significant amount of sequence mismatch (112bp; indicated by black hash marks) between the sequenced insert and the OsHV-1 ORF117 genomic sequence from GenBank, at the 5′ end of the insert.

Will pass this info along to Carolyn and Tim to see how they want to proceed.

Samples Received – White Abalone DNA from CDFW Shellfish Health Lab

Received white abalone (Haliotis sorenseni) DNA extracted from digestive gland, post-esophagus, and feces from Jim Moore and Blythe Marshman at the California Dept. of Fish & Wildlife.

These are intended for qPCR to assess presence of the RLOv.

Samples were stored in the big -20C in FSH 240.

 

Samples Received – Green Abalone Slides

Received green abalone (Haliotis fulgens) slides from Fiorenza Micheli (Hopkins Marine Station, Stanford University). These are related to the tissue she sent to us on 20160519.

Unfortunately, the writing on the casettes that were submitted to DPMG came off during processing (see note on form below). As such, the facility was unable to identify which slides came from which histology blocks. Not sure what we’ll be able to do with these. The box of slides has been stored in FSH 236 with other slide boxes.