Author Archives: kubu4

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

Sample information is in this spreadsheet (Google Sheet): ava_abalone_master_extraction_list

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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.

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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

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Project Progress – Olympia Oyster Genome Assemblies by Sean Bennett

Here’s a brief overview of what Sean has done with the Oly genome assembly front.

Metassembler

  • Assemble his BGI assembly and Platanus assembly? Confusing terms here; not sure what he means.
  • Failed due to 32-bit vs. 64-bit installation of MUMmer. He didn’t have the chance to re-compile MUMmer as 64-bit. However, a recent MUMmer announcement suggests that MUMmer can now handle genomes of unlimited size.
  • I believe he was planning on using (or was using?) GARM, which relies upon MUMmer and may also include a version of MUMmer (outdated version that led to Sean’s error message?).
  • Notebook entry

Canu

Redundans

Platanus

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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.

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Plasmid Isolation – pCR2.1/OsHv-1_ORF117 Miniprep

The last run at this failed, but I think that was due to old ampicillin stocks; leading to no selective pressure for transformants that actually contained plasmid.

I’ve since remedied that.

Grew up 5mL of culture from the only two transformants in 1xLB + 100ug/mL of (fresh!) ampicillin @ 37C on a rocking platform in a 15mL conical over night (~18hrs).

Isolated plasmid DNA from the entire 3mL of culture (repeated pelleting of bacteria in the same 1.5mL snap cap tube) using the QIAprep Spin Miniprep Kit, according to their protocol.

Eluted DNA with 50uL of EB Buffer.

Quantified on the Roberts Lab Qubit 3.0 using the dsDNA BR Kit (broad range) and 10uL of sample.

Results:

Quantification (Google Sheet): 20170817_quantification_oshv_orf117_plasmid

Colony 1 – 130ng/uL
Colony 2 – 148ng/uL

Yields look perfect. Will submit for sequencing at Genewiz (they need 10uL of ~50ng/uL DNA) and see what we have here…

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PCR – pCR2.1/OsHV-1_ORF117 Colony Screens

Performed PCR with M13 vector primers on the two colonies that grew from yesterday’s transformation.

Master mix calcs:

2x Apex Red Master PCR Mix: 33uL
M13 forward: 1.5uL
M13 reverse: 1.5uL
H2O: 29.7uL

Added 20uL to each PCR tube (0.2mL PCR strip tubes).

Bacteria was collected from each colony with a sterile 10uL pipet tip, which was used to streak on a separate LB Amp100 plate and then introduce bacteria to the appropriate PCR tube.

Cycling params (PTC-200 MJ Research):

1 cycle:

95C – 10mins

30 cycles:

95C – 15s
55C – 15s
72C – 90s

1 cycle:

72C – 10mins

PCR reactions were run on a 1% agarose 1xTBE gel + EtBr.

5uL of O’GeneRuler DNA Ladder Mix was loaded for sizing.

Results:

 

 

Well, this might seem promising, due to the intensity of that band (~1000bp). A band of that size was also produced the last time, ableit with much less intensity.

The very bright, 1000bp band generated from Colonies 1 (left) and 2 (right) is not the expected size. Based on this paper (Detection of undescribed ostreid herpesvirus 1 (OsHV-1) specimens from Pacific oyster, Crassostrea gigas. Martenot et al. 2015), the insert size should be ~1300bp (Tim Green indicated he used the primers listed in the paper to clone ORF117).

However, there is a less bright band just above 1500bp. Oddly, this would be the expected size for this PCR (1300bp insert + 200bp of vector sequence from the M13 primers). The lower intensity is discouraging, though, because this indicates that M13 primers are preferentially binding whatever is producing that 1000bp band.

Regardless, I’ve already inoculated two liquid cultures to grow up over night. I’ll perform a plasmid isolation on them tomorrow morning. Hopefully they actually yield some plasmid DNA to do some work with, unlike last time.

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Transformation – pCR2.1/OsHV-1_ORF117 into One Shot Top10 Chemically Competent Cells

Yesterday’s transformation with freshly prepared ampicillin didn’t produce any transformants, suggesting the DNA concentration is too low.

Previously, I tried to elute the DNA from one of the spots Tim sent with 50uL. This volume was enough to soak the Whatman paper and produce excess liquid. In retrospect, I think the volume was too large and diluted the DNA too much (concentration wasn’t measurable via Qubit)

Today, I eluted with 25uL. Since this volume was too little to produce excess liquid, I created a spin “filter” to extract the absorbed liquid. Briefly, I punctured the top and bottom of a 0.5mL snap cap tube with an 18 gauge needle, inserted the Whatman paper disc into this tube, and then put this tube in a 2mL snap cap tube. This assembly was spun @ 18,000g RT for 3 mins.

Used 5uL of the pCR2.1/OsHV-1_ORF117 plasmid provided by Tim Green to transform a single aliquot of One Shot Top10 Chemically Competent Cells (Invitrogen), according to the “Rapid Transformation” protocol (thaw cells on ice, add DNA, incubate 5mins, plate on pre-warmed ampicillin plates).

Cells were plated on pre-warmed (37C) LB Amp100 plates.

Plates were incubated overnight at 37C.

Results:

Wow, only two colonies! Well, as they say, you only need one. Will PCR, re-streak, and inoculate 5mL liquid cultures to see if either of these colonies seem to have the insert.

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