# DNA Isolation – Ava Withering Syndrome Transmission Study Tissues

Isolated DNA from 17 red abalone digestive gland tissue samples.

Tissue was weighed, minced with a razor blade, and transferred to 2mL snap cap tube containing 1mL of InhibtEX Buffer.

DNA was extracted using the QIAmp Fast DNA Stool Mini Kit (Qiagen) following the manufacturer’s protocol with the following options:

Minced tissue was incubated at 70C O/N.

These samples were actually incubated O/N on 20171012. I dropped the rack containing these tubes after the initial incubation and these tubes popped open and spilled.

Since I used all of the tissue, I have nothing to go back to. I’ve attempted to recover as much of the remaining supernatant in each of these tubes. I brought the volume of each tube up to 600mL with Inhibitex Buffer and proceeded with the isolations.

Followed “human DNA analysis” protocol (to maximize sample recovery)
Eluted DNA with 100μL Buffer ATE

# DNA Isolation – Ava Withering Syndrome Transmission Study Tissues

Isolated DNA from 96 red abalone digestive gland tissue samples.

Tissue was weighed, minced with a razor blade, and transferred to 2mL snap cap tube containing 1mL of InhibtEX Buffer.

DNA was extracted using the QIAmp Fast DNA Stool Mini Kit (Qiagen) following the manufacturer’s protocol with the following options:

Minced tissue was incubated at 70C O/N
Followed “human DNA analysis” protocol (to maximize sample recovery)
Eluted DNA with 100μL Buffer ATE

# DNA Isolation – Ava Withering Syndrome Transmission Study Tissues

Isolated DNA from 144 red abalone digestive gland tissue samples.

Tissue was weighed, minced with a razor blade, and transferred to 2mL snap cap tube containing 1mL of InhibtEX Buffer.

DNA was extracted using the QIAmp Fast DNA Stool Mini Kit (Qiagen) following the manufacturer’s protocol with the following options:

Minced tissue was incubated at 70C O/N
Followed “human DNA analysis” protocol (to maximize sample recovery)
Eluted DNA with 100μL Buffer ATE

# DNA Isolation – Ava Withering Syndrome Transmission Study Tissues

Isolated DNA from 58 red abalone digestive gland tissue samples.

Tissue was weighed, minced with a razor blade, and transferred to 2mL snap cap tube containing 1mL of InhibtEX Buffer.

DNA was extracted using the QIAmp Fast DNA Stool Mini Kit (Qiagen) following the manufacturer’s protocol with the following options:

• Minced tissue was incubated at 70C O/N
• Followed “human DNA analysis” protocol (to maximize sample recovery)
• Eluted DNA with 100μL Buffer ATE

# DNA Isolation – Ava Withering Syndrome Transmission Study Tissues

Isolated DNA from 26 red abalone digestive gland tissue samples.

Tissue was weighed, minced with a razor blade, and transferred to 2mL snap cap tube containing 1mL of InhibtEX Buffer.

DNA was extracted using the QIAmp Fast DNA Stool Mini Kit (Qiagen) following the manufacturer’s protocol with the following options:

• Minced tissue was incubated at 70C O/N
• Followed “human DNA analysis” protocol (to maximize sample recovery)
• Eluted DNA with 100μL Buffer ATE

# 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.

# Data Aggregation – Ava’s Complete Sample List

I received Ava’s master sheet of all the samples she collected for this project. I needed to aggregate a full list of the samples I’ve previously extracted DNA from, so that I can compare to her master sample list and generate a list of the remaining samples that I need to extract DNA from..

Here are the files I needed to work with (Google Sheets):

The files required multiple formatting steps in order to produce accession numbers that were formatted in the same fashion across all three sheets. This was needed in order to be able to successfully merge all of the sheets into a single sheet containing all of the data, which will make it easy to sort, and generate a list of samples that need to be extracted.

Text file manipulations were performed in a Jupyter notebook, which is linked below. All files were downloaded from Google Sheets as tab-delimited files prior to working on them.

Jupyter Notebook file: 20170831_ava_ab_samples_aggregation.ipynb
Jupter Notebook on NBviewer: 20170831_ava_ab_samples_aggregation.ipynb

Now that we have the tables formatted, we can use the accession number as a common field by which to combine the two tables. This will allow easy sorting and identification of the remaining samples that I need to extract. I’ll do this by using SQLite3.

Use SQLite3 (in Linux Ubuntu):

Change to directory containing files:

cd ~/Dropbox/Sam\ Friedman\ Lab/tmp

Start SQLite3:

sqlite3

Set field separator as tab-delimited:

.separator "\t"

Create databases by importing files and providing a name for corresponding databases:

.import ava_master_ab_list_formatted.tsv master_list
.import Ava_WS_Transmission_DNA_Extractions_all.tsv extracted_list

Set output display mode to tabs:

.mode tabs

Set output display to include column headers:

.headers on

Set the output to write to a file instead of the screen:

.output 20170905_master_extraction_list.tsv

SELECT statement to combine the two tables:

SELECT * FROM (SELECT * FROM master_list UNION ALL SELECT * FROM extracted_list) s GROUP BY accession_number ORDER BY accession_number;

The SELECT statement above works in the following fashion:

Uses a sub-query (contained in the parentheses) that combines all of the rows in both tables and creates an intermediate table (that’s the s after the sub-query). Then, all of the columns in that intermediate table are selected by the initial SELECT * FROM and organized by the GROUP BY clause (which combines any rows with identical values in the accession_number column) and then sorts them with the ORDER BY clause.

After that’s finished, we want to reset the output to the screen so we don’t overwrite our file:

.output stdout

The output file is here (Google Sheet): ava_abalone_master_extraction_list

# 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.