Ecology of Infectious Marine Disease Lab Notebook

Week 1

7/23/12

After a day of orientation, we divided up into lab groups based roughly on our experience with different techniques/fields. I'm with Annie and Courtney - we're the only lab group that has to look across the whole lab to look out the window and see the ocean.

7/24/12

Following a lecture on general invertebrate anatomy (of gastropods, molluscs, crustaceans), we spent the morning's lab working on dissections.
DSC_0363.JPG

7/25/12

DSC_0380.JPG
Armina digestive gland being placed into a histology cassette

7/26/12

DNA Extractions from Armina Tissue
DNA extraction from the tissue samples taken yesterday from Armina, accession number 12-1-6 for the sample I extracted. Followed protocol exactly but at the point of pipetting the sample into the spin column, made the following mistake. Instead of pipetting the full sample into the column, took 200 uL into one spin column, so did another column with the rest of the sample. Used the same amount of buffer for intermediate steps with the column and then combined DNA extractions after elution. DNA extraction stored in the freezer/fridge (?) until the afternoon, at which time the following PCR was run.
PCR Master Mix Calculations (originally done in excel)
Two Primer Sets (generic) Master Mix
  1. Reactions =
 8

uL/Reaction
 uL/8 Reactions
Immomix 2x
 12.5
 100

BSA (10 mg/ml)
 1.5
 12

Fwd primer (10 um)
 0.8
 6.4

Rev primer (10 um)
 0.8
 6.4

sH2O
 7.4
 59.2

Template
 2
 16
 23
Total
 25
 200






WS-RLO cPCR Master Mix
  1. Reactions =
 8
Reagent
 [Stock]
 [End]
 Per reaction (uL)
 Per 8 reactions (uL)
5 X Buffer
 5 X
 1 X
 4
 32
MgCl2
 25 mM
 1.5 mM
 1.2
 9.6
BSA
 10 mg/ml
 400 ng/ml
 0.8
 6.4
H2O

11.08
 88.64
dNTP's
 10 mM
 200 uM
 0.4
 3.2
RA 3-6
 100 pmol/ml
 0.5 uM
 0.1
 0.8
RA 5-1
 100 pmol/ml
 0.5 uM
 0.1
 0.8
Taq
 5 U/ul
 1.6 U
 0.32
 2.56
Template

2
 16
Total

20
 160
PCR Lane Assignments
1
 2
 3
 4
 5
 6
Negative Control
 Blank Control
 CC 12-1-6
 GS 12-1-6
 2010 Sample (Annie)
 Positive Control
PCRs were run with three sets of primers: 2 sets of 'generic' primers (universal bacterial EUB A/B, Ehrlichia - EHR16s) and one WS-RLO specific set (RA 3-6/RA 5-1) developed by the Friedman lab for identifying WS-RLO.
Additional Armina Dissection with Maya

7/27/12

Gel of Yesterday's PCR
Instead of using ethidium bromide (a very bad carcinogen) we will be using Cybersafe (a not so bad carcinogen) to stain the gel, intercalate with the DNA and visualize the amplified product.

Visualization of gel using transilluminator. Gel product shown in the image below, with primer sets and samples labeled. No positive control showed up for the WS-RLO primers, but both the universal bacterial and ehrlichia primers showed a band at the positive control. Universal bacterial primers showed a band for the 12-1-6 CSC, which was a piece of digestive gland tissue isolated from an Armina dissected in 2012. Other bands that are faint and don't line up with the positive control are likely primer dimers.
7-27-12 Armina Gel.jpg

Week 2

8/3/12


Team Crosson Protocol for Inoculations (written by Sonia)
Day 1-2
Day 3
Day 4-6

Team Harvell Protocol for Inoculations (written by Sonia, adapted from [Muhlstein et al. 1988])
Day 1

Day 2
Day 3

Day 4 +

Week 3

8/3/12



Week 4

8/13/12

PM Lab: Learning how to play with BLAST and doing things with "My favorite gene"

Terminal (how to access BLAST through the terminal)

How to run an actual BLAST from the command line for the QPX data:
Started working on my favorite gene project. I've chosen to work on amoA, which is a subunit of an enzyme responsible for early steps in nitrification. See the progress at the google doc as work happens: https://docs.google.com/document/pub?id=1boTFn-f97XuN8PfYrzrqMlQrZcbe6A9dyKa3BUeb-Mw

8/14/12

AM Lab: QPX Data Analysis

We received the first batch of data off the Illumina Hi-Seq machine.
With regard to QPX- three libraries were sequenced
- DNA
- mRNA with culture grown at 10C
- mRNA with culture grown at 21C

You can see the raw data (or download) from the DNA library @
http://aquacul4.fish.washington.edu/~steven/armina/filtered_QPX_DNA_GTGAAA_L001_R1.fastq.gz

de novo assembly using CLC workbench. There are other types of software to do this with, including Trinity (?), Geneious, Newbler. Assembly uses the "Greedy algorithm", and we can specify the stringency requirements depending on species, HTS machine. Can also use paired-end sequencing to overlay a spatial component onto sequence data, placing the short reads in relationship to each other.

Map reads back onto the assembled contigs to generate an RNA-seq file. Set parameters s.t. number of mismatches is 2, and specify the number of times a read can go to multiple contigs. Tab delimited file will return data including coverage, gene length, etc. and RPKM (which normalizes for library size:
"Feature ID"    "Expression values"    "Gene length"    "Unique gene reads"    "Total gene reads"    "RPKM"
Use CLC to compare the two QPX treatment libraries in an "experiment" to generate a file that can be cut down into fold-change difference between the two: CLC Fold-Change Results

Proceed by BLASTing the transcriptome (FULL transcriptome) that was assembled from the 10 and 21 degree C libraries. So, to do this we are going to do the following.

PM Lab:
Upload "fold-change" file to Galaxy. Download the transcriptome->SwissProt mapping file.

Join tables from transcriptome, gene expression change table,

With the GO Slim Association Table

Import file, TAB delimit, join with SPID and description, join with Swiss + Associations, join with

8/15/12

AM Lab:

Enrichment Analysis

Gene Set enrichment analysis on DAVID


8/16/12

Metagenomic Analyses

PM Lab

8/17/12

QPX Transcriptome to Genome Blast, using genome contigs with 1000 bp length

BLAST2GFF

8/18/12

Why share data?

8/19/12

BLAST2GFF trying to map nonsynonymous SNP mutations to the QPX genome

Once the GFF file was generated, it was uploaded to Galaxy, specifying the file format to be GFF. Once uploaded, the file was converted to a BED format and uploaded to the QPX genome track.

8/21/12



In GALAXY


8/21/12