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.

Plasmid Curve – RLP Plasmid Size Determination

After looking through Nate’s notebook and aligning the withering syndrome primers and probe to their source sequence (GenBank Accession AF133090; see below) I have discovered a few things:

  1. The vector that was used for cloning was Invitrogen’s PCR 4-TOPO (3956bp). This was found in Nate’s notebook #1 WS qPCR on page 3.

  2. The insert was generated with the primers RA 5-1 and RA 3-6. The resulting fragment size is 158bp. However, Nate’s notebook lists the size as 176bp.

  3. The size of the vector plus the insert should be 4114bp. However, since Nate used the incorrect insert size, he calculated the combine vector+insert size as 4133bp.

  4. Although this doesn’t seem to have much of an effect on the withering syndrome qPCR assay (based on the assay working for years prior to our current issues), the WSN1R primer sequence actually lies partially outside of the cloned region of AF133090. See the alignment below.

Made the dilutions for the RLP standard curve, based on a size of 4114bp. The calculations are here.

20120406 – UPDATE: Carolyn has just remembered that there are multiple, different versions of RLP cloned into a variety of vectors. After discussion with Carolyn and Robyn, Carolyn definitively determined that Lisa and I had not been using the correct clones with the correct RLP insert! The correct vector/insert should be p16RK3-C with the entire AF133090 sequence insert.