Category Archives: Lineage-specific DNA methylation patterns in developing oysters

Lineage-specific DNA methylation patterns in developing oysters

Goals – May 2015

Here are the things I plan to tackle throughout the month of May:

Geoduck Reproductive Development Transcriptomics

My primary goal for this project is to successfully isolate RNA from the remaining, troublesome paraffin blocks that have yet to yield any usable RNA. The next approach to obtain usable quantities of RNA is to directly gouge tissue from the blocks instead of sectioning the blocks (as recommended in the PAXgene Tissue RNA Kit protocol). Hopefully this approach will eliminate excess paraffin, while increasing the amount of input tissue. Once I have RNA from the entire suite of samples, I’ll check the RNA integrity via Bioanalyzer and then we’ll decide on a facility to use for high-throughput sequencing.

 

BS-Seq Illumina Data Assembly/Mapping

Currently, there are two projects that we have performed BS-Seq with (Crassostrea gigas larvae OA (2011) bisulfite sequencing and LSU C.virginica Oil Spill MBD BS Sequencing) and we’re struggling to align sequences to the C.gigas genome. Granted, the LSU samples are C.virginica, but the C.gigas larvae libraries are not aligning to the C.gigas genome via standard BLASTn or using a dedicated bisulfite mapper (e.g. BS-Map). I’m currently BLASTing a de-novo assembly of the C.gigas larvae OA 400ppm sequencing that Steven made against the NCBI nt DB in an attempt to assess the taxonomic distribution of the sequences we received back. I’ll also try using a different bisulfite mapper, bismark, that Mackenzie Gavery has previously used and has had better results with than BS-Map.

 

C.gigas Heat Stress MeDIP/BS-Seq

As part of Claire’s project, there’s still some BS-Seq data that would be nice to have to complement the data she generated via microarray. It would be nice to make a decision about how to proceed with the samples. However, part of our decision on how to proceed is governed by the results we get from the two projects above. Why do those two projects impact the decision(s) regarding this project? They impact this project because in the two projects above, we produced our own BS-Seq libraries. This is extremely cost effective. However, if we can’t obtain usable data from doing the library preps in-house, then that means we have to use an external service provider. Using an external company to do this is significantly more expensive. Additionally, not all companies can perform bisulfite treatment, which limits our choices (and, in turn, pricing options) on where to go for sequencing.

 

Miscellany

When I have some down time, I’ll continue working on migrating my Wikispaces notebook to this notebook. I only have one year left to go and it’d be great is all my notebook entries were here so they’d all be tagged/categorized and, thus, be more searchable. I’d also like to work on adding README files to our plethora of electronic data folders. Having these in place will greatly facilitate the ability of people to quickly and more easily figure out what these folders contain, file formats within those folders, etc. I also have a few computing tips/tricks that I’d like to add to our Github “Code” page. Oh, although this isn’t really lab related, I was asked to teach the Unix shell lesson (or, at least, part of it) at the next Software Carpentry Workshop that Ben Marwick is setting up at UW in early June. So, I’m thinking that I’ll try to incorporate some of the data handling stuff I’ve been tackling in lab in to the lesson I end up teaching. Additionally, going through the Software Carpentry materials will help reinforce some of the “fundamental” tasks that I can do with the shell (like find, cut and grep).

In the lab, I plan on sealing up our nearly overflowing “Broken Glass” box and establishing a new one. I need to autoclave, and dispose of, a couple of very full biohazard bags. I’m also going to vow that I will get Jonathan to finally obtain a successful PCR from his sea pen RNA.

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DNA Quantification – Claire’s C.gigas Sheared DNA

In an attempt to obtain the most accurate measurement of Claire’s sheared, heat shock mantle DNA, I quantified the samples using a third method: fluorescence.

Samples were quantified using the Quant-It DNA BR Kit (Life Technologies/Invitrogen) according the manufacturer’s protocol. Standards were run in triplicate. Due to low sample volumes, only 1μL of each sample was used and was not replicated.

Plate was read on a Perkin Elmer plate reader using the Wallac software. The plate was measured three times, with each well measured for a one second duration on each read.

 

Results:

Spreadsheet: 20150303_gigasHSshearedDNApico

 

 

Comparison of NanoDrop1000, Bioanalyzer, and fluorescence measurements:

Sample NanoDrop (ng/μL) Bioanalyzer (ng/μL) Fluorescence (ng/μL)
2M sheared 48.03 16.28 4.91
4M sheared 190.96 58.52 48.10
6M sheared 141.56 42.98 28.42
2MHS sheared 221.93 32.45 13.48
4MHS sheared 257.48 43.82 11.75
6MHS sheared 202.02 51.12 8.97

 

Not entirely surprising, but the fluorescence method is clearly the most conservative measurement of the three methods. However, I do find the difference between the Bioanalyzer and fluorescence measurements very surprising. I suspected the Bioanalyzer would underestimate the concentrations because I actively selected which peak regions to measure, possibly leaving out some aspect of the sample.

Regardless, will use the most conservative measurements (fluorescence) for decision making.

With our yields, we have insufficient DNA to conduct MeDIP and then subsequent bisulfite conversion and library prep on our own. The recovery from the MeDIP will result in too little input DNA for bisulfite conversion and, in turn, library prep.

However, we do have sufficient quantities of starting DNA (>200ng) for Epigentek’s MeDIP Methyl-seq. I have contacted Epigentek to see if their procedure includes bisulfite conversion after MeDIP (which the website workflow suggests that it does not).

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DNA Quantification – Claire’s Sheared C.gigas Mantle Heat Shock Samples

I previously checked Claire’s sheared DNA on the Bioanalyzer to verify the fragment size and to quantify the samples. Looking at her notebook, her numbers differ greatly from the Bioanalyzer, possibly due to the fact that the DNA1000 assay chip used only measures DNA fragments up to 1000bp in size. If her shearing was incomplete, then there would be DNA fragments larger than 1000bp that wouldn’t have been measured by the Bioanalyzer. So, I decided to quantify the samples on the NanoDrop1000 (ThermoFisher) again.

 

Results:

Spreadsheet: 20150226_Claire_sheared_Emma_1000ppm_OD260s

 

 

 

Comparison of NanoDrop1000 and Bioanalyzer measurements.

Sample NanoDrop (ng/μL) Bioanalyzer (ng/μL)
2M sheared 48.03 16.28
4M sheared 190.96 58.52
6M sheared 141.56 42.98
2MHS sheared 221.93 32.45
4MHS sheared 257.48 43.82
6MHS sheared 202.02 51.12

The NanoDrop is known to overestimate sample quantities due to the indiscriminate nature of UV spectrophotometry and that could be the reason for the large discrepancy between the two measurements (i.e. RNA carryover may lead to overestimation). As such, I’ll quantify the samples using a fluorescence-based assay for double stranded DNA tomorrow in hopes of getting the most accurate measurement.

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Bioanalyzer – C.gigas Sheared DNA from 20140108

To complement MBD ChiP-seq data and RNA-seq data that we have from this experiment, we want to generate, at a minimum, some BS-seq data from the same C.gigas individuals used for the other aspects of this experiment.  Claire had previously isolated DNA and sheared the DNA on 20140108. If possible, we’d like to perform MBD enrichment, but the current quantities of DNA may prevent us from this.

To quantify the DNA and evaluate the shearing profile, I ran 1μL of each of the following mantle pre-/post-heat shock samples on a DNA 1000 chip (Agilent) on the Agilent 2100 Bioanalyzer. in the Seeb Lab:

M = mantle
HS = heat shocked

  • 2M sheared
  • 4M sheared
  • 6M sheared
  • 2M HS sheared
  • 4M HS sheared
  • 6M HS sheared

Results:

Bioanalyzer Data File (XAD): 2100_expert_DNA_1000_DE72902486_2015-02-19_11-32-35(2).xad

 

Electropherograms

2100 Bioanalyzer electropherograms of Claire’s sheared C.gigas DNA.

 

Spreadsheet: 2100 expert_DNA 1000_DE72902486_2015-02-19_11-32-35_Results_001

 

Claire’s notebook entry doesn’t ever specify what her target shear size was, but the Bioanalyzer analysis suggests an average size of ~500bp.

Also interesting to note is that Claire’s sample concentrations (as measured on the NanoDrop1000) are significantly greater than what is calculated by the Bioanalyzer. Since the Bioanalyzer chip used (DNA1000) only goes to 1000bp, is it possible the differences in concentrations is due to incomplete shearing of the samples (e.g. a significant portion of the DNA is >1000bp in size and thus not factored in to the Bioanlyzer concentrations calculations)?

Will check sample volumes and determine total amount of remaining DNA for each sample and then assess how to proceed next (i.e. MBD or just BS-seq).

UPDATE 20150226:

Sample volumes were measured and total quantity (ng) of DNA in each sample were added to the spreadsheet above.

Based on the quantities of DNA we have for each sample, will discuss sequencing options (e.g. MBD or not, self-prepare libraries or not, etc) with Steven.

 

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