Today I met with Steven and Sam to make a new plan for the crab RNA problem. We’re going to use the lyophilizer (freeze drying machine that goes below -80˚C) on 8 new pelleted hemolymph samples from Day 26; 8 supernatant samples that correspond to those 8 pelleted; and a pooled sample of all the ones Sam processed using the Qiagen RNeasy Kit that had “out of range” Qubit results. This will have to be post-poned for a bit becuase the lyophilizer is currently being fixed… waiting for an ETA on that. In regards to Skyline, I re-visited what I did before I left for vacation and went through all the files used and settings chosen. I put all of the information in a new GitHub issue per Steven’s request.
Sam processed the 40 samples that I picked out from Day 26 (samples taken in triplicate) using the Qiagen RNeasy Kit. 15 of the 40 had detectable RNA with the Qubit. Here is a link to his notebook post: RNA Isolation & Quantification – Tanner Crab Hemolymph.
Steven, Sam, and I talked a little before lab meeting to come up with a new game plan. I picked out 8 more samples (2 from each treatment group from day 26 except for the warm). I pulled out the pelleted hemolymph as well as the supernatant (RNAlater) that may have some cells in it.
I also pooled together all of the samples that Sam processed that had “out of range” Qubit results.
THe plan (once the Lyophilizer is fixed/ if it is fixed) is to put the samples in the lyophilizer (freeze dryer) overnight, then try out the Tri-reagent protocol on the 8 pelleted and 8 supernatant samples. I will re-quanitifiy the pooled sample using the QUbit.
Based on what happens with that, we’ll decide what else to do going forward. But we want to send off a tube for sequencing ASAP so that we can get things moving.
I made a GitHub issue (#341) detailing the issues I’ve had since using Walnut to make the new BLIB file. Assigned Emma to the issue and am awaiting her response.
Read The epigenetic landscape of transgenerational acclimation to ocean warming to get an idea for potential analysis methods. The authors used different functions in methylKit to obtain DMRs:
"Briefly, the ‘methRead’ function of methylKit reads the mapping results with 10 reads per cytosine as a minimum coverage threshold. High coverage bases (99.9%) were filtered to exclude potential PCR bias and then normalized using ‘filterByCoverage’ and ‘normalizeCoverage’ functions, respectively. Genomic regions were categorized as CpG island, CpG shore, promoter, 5′ untranslated region (UTR), exon, introns, 3′ UTR and repeats. Methylated or unmethylated cytosines in each genomic region were summed for each sample by the ‘regionCounts’ function of methylKit. The P values of methylation differences for each region between two samples were calculated using a chi-squared test in the ‘calculateDiffMeth’ function."
They also generated heatmaps with DMR data, which would be useful in my case as well.
In BEDtools, they used the closest function to pair DMRs and genes:
"The closest gene to a DMR on the same scaffold was identified using ‘closest’ from BEDTools v. 2.2339. This resulted in 1,563 genes from 2,078 CpG DMRs, while 115 DMRs were on scaffolds without annotated genes (Supplementary Table 4)."
Because they paired gene expression data with epigenetic data, they did not do any gene enrichment. I’ll need to refer to Emma’s geoduck paper for those methods.
Protocol test: Round 3
TL;DR Nothing really works.
- Tissue Tearor at setting 2 for 20 s (Note: Laura said she used the Tissue Tearor at setting 15 in this lab notebook. She must have used one different than what’s in FSH 213, because there was no setting 15 on the one I used).
- Glass beads + vortexing for 2 minutes
- Glass beads + vortexing for 5 minutes
- Glass beads + vortexing for 10 minutes
Figure 1. Tissue Tearor used for protocol.
- My plan was to take 0.08 g of tissue from 3 separate tissue samples, and split it amongst the methods listed above. Tube labels consisted of a tissue marker (T1, T2, or T3), and the method (TT for Tissue Tearor, V2 for 2 minute vortex, V5 for 5 minute vortex, and V10 for 10 minute vortex). However, the tissues themselves had varying thickness and sizes, so I used 2 tissues from the same block for each tissue marker. Here’s how the tissue was split up. T1 and T2 were taken from the 1 notch block, and T3 was taken from the 5 notch block.
Figure 2. 5 notches (left) and 1 notch (right)
Figures 3-4. Division of tissues for histology.
- I transfered the extracted tissue to the tube first, then mashed it in the tube so get it into finer pieces. While transferring, I accidentally added T3-V5 tissue to T3-V2. I took the big chunks that weren’t pressed against the tube walls out and put it in T3-V5.
- In between tissues, I dipped my scalpel in a 20% bleach solution, then washed with DI water
- While carving out tissue, I avoided tissue that had histology ink on it. However, after the xylene addition, there was a blue histology ink hue in tube T1-TT.
- Step 9: I incubated the tubes for 10 minutes at room temperature, and 13 minutes at 37ºC.
- Step 12-13: The adapter for the vortex didn’t work, so I had to hold the tubes on the vortex manually. Either I now have extremely great circulation in my hands, or I did something very bad.
- Step 12-13: In between samples while using the Tissue Tearor, I cleaned the tip in ethanol for 15 s, then 20 s in DI water
- Step 14: I got the adapter to work by holding it in place with my hands! Better than holding three tubes on the vortex for 10 minutes continuously.
- Step 16: There was no gelatinous pellet after incubation and vortexing, so I think the Proteinkinase K digestion went well
- Step 17: Incubated the tubes 1 extra minute while waiting for the thermomixer to reach 80ºC
- Step 28: I multipied the recipe from previous days by 5. I mixed 3283.5 µL of the buffer with 16.5 µL of dye. S1 = 181.41, S2 = 22377.75.
Table 1. Tube number, initial sample added, and DNA concentration. All of these concentrations are too low for downstream appliciations, and there does not seem to be any pattern regarding method and yield. T2-V2 yield was too low for the Qubit to measure.
|Tube||Initial Mass (g)||DNA Concentration (ng/µL)|
Seeing how these changes to the protocol are not working and I have to return the thermomixer first thing Monday morning, I think I should move on to either 1) RNA extractions from the histology cassettes or 2) DNA extractions from ctenida, mantle, or adductor tissue. I updated this issue for a response.
[sr320@mox2 jobs]$ cat 0809_1500.sh #!/bin/bash ## Job Name #SBATCH --job-name=angsd-05 ## Allocation Definition #SBATCH --account=srlab #SBATCH --partition=srlab ## Resources ## Nodes (We only get 1, so this is fixed) #SBATCH --nodes=1 ## Walltime (days-hours:minutes:seconds format) #SBATCH --time=10-100:00:00 ## Memory per node #SBATCH --mem=100G #SBATCH --mail-type=ALL #SBATCH --email@example.com ## Specify the working directory for this job #SBATCH --workdir=/gscratch/srlab/sr320/analyses/0809b source /gscratch/srlab/programs/scripts/paths.sh /gscratch/srlab/sr320/programs/angsd/angsd -bam /gscratch/srlab/sr320/data/cw/all_bam.bamlist -out Genotypes_parentage2 -GL 1 -doMaf 1 -doMajorMinor 1 -minMaf 0.1 -SNP_pval 1e-6 -minInd 525 -minQ 20 -P 28 -doGeno 2 -doPost 1 -postCutoff 0.95 -doCounts 1 -geno_minDepth 5[sr320@mox2 jobs]$
[sr320@mox2 jobs]$ cat 0809_1300.sh #!/bin/bash ## Job Name #SBATCH --job-name=angsd-05 ## Allocation Definition #SBATCH --account=srlab #SBATCH --partition=srlab ## Resources ## Nodes (We only get 1, so this is fixed) #SBATCH --nodes=1 ## Walltime (days-hours:minutes:seconds format) #SBATCH --time=10-100:00:00 ## Memory per node #SBATCH --mem=100G #SBATCH --mail-type=ALL #SBATCH --firstname.lastname@example.org ## Specify the working directory for this job #SBATCH --workdir=/gscratch/srlab/sr320/analyses/0809 source /gscratch/srlab/programs/scripts/paths.sh /gscratch/srlab/sr320/programs/angsd/angsd \ -bam /gscratch/srlab/sr320/data/cw/all_bam.bamlist \ -out Association_test2 \ -doAsso 1 \ -yBin /gscratch/srlab/sr320/data/cw/YBin_file \ -GL 1 \ -doMaf 1 \ -doMajorMinor 1 \ -minMaf 0.01 \ -SNP_pval 1e-6 \ -minInd 468 \ -minQ 20 \ -doGlf 3 \ -P 28
Received the data from the geoduck metagenome libraries that I prepared and were sequenced at the Northwest Genomics Center at UW on the HiSeqX (Illumina) – PE 151bp.
FastQ files are being transferred to owl/nightingales/P_generosa.
These aren’t geoduck sequences, but they are part of a geoduck project. Maybe I should establish a metagenomics directory under
Will verifiy md5 checksums and update readme file once the transfer is complete.