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