Category Archives: 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.



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.

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.



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).

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.



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.

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


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



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.


DNA Isolation – Claire’s C.gigas Female Gonad for Illumina Bisulfite Sequencing

Due to poor “tag counts” from the initial sequencing (DATE) and the re-sequencing (20131127) of this sample, the HTGU facility has concluded that the library is probably at fault. They will make a new library and do a quality control run on the new library. However, they have insufficient gDNA left to make a new library.

Isolated gDNA from Claire’s sample following the DNAzol protocol.

Transferred ~300uL of female C.gigas gonad from the source tube (ethanol-preserved) to a clean tube. Pelleted gonadal material by spinning 10,000g, 30seconds, @ RT. Decanted residual ethanol. Resuspended tissue in 500uL of DNAzol + 100ug of Proteinase K (Fermentas; 18.5mg/mL). Incubated on a rotator for ~6hrs. Proceeded according to DNAzol protocol. Resuspended final pellet in 100uL of Elution Buffer (Qiagen; EB). After resuspension, pelleted remaining debris 16,000g, 30seconds, @ RT. Transferred supernatant to clean tube and quantified on NanoDrop 1000.

CgF – 403.2ng/uL

Will bring tube to sequencing facility tomorrow morning.

DNA Isolation – Claire’s C.gigas Female Gonad

Trying this sample again(!!), but will now use TE for pellet resuspension to prevent sample degradation. Incubated sample RT on rotator in 500uL of DNazol + 2.7uL of Proteinase K (Fermentas; Stock 18.5mg/mL) for 5hrs. Added additional 500uL of DNazol, mixed gently and followed DNazol manufacturer’s protocol. Performed first pellet was with 70% DNazol/ 30% EtOH solution. Resuspended pellet in 200uL of TE and spec’d on NanoDrop1000.


Yield is good. 260/280 value is good. 260/230 value is poor. Will run on gel to evaluate integrity.

Loaded 10uL (~830ng) on 1.0% agarose 1x modified TAE gel stained with EtBr.

Gel Loading Guide:

Lane 1 – Hyperladder I (Bioline)

Lane 2 – C.gigas female gonad gDNA (CgF)

Well, this certainly looks much better than previous preparations, in that there is an obvious high molecular weight band present (previously, this had been absent). The low molecular weight bands/smears are possibly RNA carryover and/or degraded DNA. Will discuss with Steven and then, most likely, bring downtown for Illumina sequencing.

UPDATE 20140508: Downtown sequencing facility says there’s only ~800ng of DNA! This is a far cry from the minimum amount needed for sequencing (6ug). Looking at the gel above and comparing sample band intensity to the ladder band intensities suggests that the downtown sequencing facility is correct. I loaded 10uL of DNA on the gel and the intensity of the high molecular weight band is similar to the 400bp band intensity. This corresponds to 40ng of DNA. That means the CgF gDNA band is 40ng/10uL = 4ng/uL. I resuspended the gDNA pellet in 200uL of TE, so 200uL x 4ng/uL = 800ng; exactly what the sequencing facility says they measured…

I’m not entirely sure what is happening here. Until very recently, there were almost never such egregious differences between the NanoDrop measurements and what they were measuring downtown at the sequencing facility. It seems as though they have changed the way they quantify samples (possibly using an Agilent Bioanalyzer instead of the Life Technologies Qubit fluorometer?), but this doesn’t mean their measurements are incorrect. However, I’m starting to suspect that the reason the initial sequencing of this sample was due to an overestimation of the quantity of input DNA (since I believe they were still using the fluorometer back then).

As such, it’s become clear that C.gigas gonad samples seem to yield poor quantities of gDNA, relative to the amount of input material. Additionally, there may be insufficient sample left to generate a useable quantity of gDNA to complete this resequencing effort.

DNA Isolation – Test Sample

Due to the recent poor quality gDNA that has been isolated from C.gigas gonad, I decided to do a quick test using TE for DNA pellet resuspension in hopes that old Buffer EB (Qiagen) or old nuclease-free H2O (Promega) are to blame for the apparent, rapid degradation that I’ve experienced.

Isolated gDNA from a C.gigas female gonad sample (EV2 141 go) provided by Mac. Isolated gDNA using DNazol (Molecular Research Center):

  1. Incubated ~25mg of tissue O/N @ RT in 500uL of DNazol + 100ug/mL Proteniase K (2.7uL of 18.5mg/mL Fermentas stock) on rotator.

  2. Added additional 500uL of DNazol and briefly disrupted remaining tissue with a few pipette strokes.

  3. Pelleted debris by spinning 10mins, 10,000g @ RT.

  4. Transferred supe to new tube and repeated Steps 3 & 4 one time.

  5. Added 500uL of 100% EtOH; mixed by inversion.

NOTE: Despite initial appearance of white cloudy appearance after EtOH addition, cloudiness dispersed upon inversion and no visible DNA strands were present

  1. Pelleted DNA by spinning 5000g 5mins @ RT.

  2. Removed supe and washed pellet with 1mL of a 70% DNazol+30% EtOH solution.

  3. Removed supe and washed pellet with 1mL 70% EtOH.

  4. Repeated Step 8 two times.

  5. Discarded supe, quick spun tube to pool residual EtOH. Removed all residual EtOH.

  6. Resuspended in 200uL of TE (pH = 8.0) and incubated at RT for 5mins.

  7. Pelleted insoluble material 12,000g 10mins @ RT.

  8. Transferred supe to clean tube.

  9. Spec’d on NanoDrop1000.

  10. Ran ~500ng on 1.0% agaroase 1x modified TAE gel to evaluate integrity.


260/280 value looks excellent, but, as always seems to be the case with DNazol/TriReagent, the 260/230 value looks crappy. Will investigate gDNA integrity on agarose gel.

Gel Loading:

Lane 1 – Hyperladder I (Bioline)

Lane 2 – EV2 141 go C.gigas female gonad gDNA

Well, look at that! A nice, clear, high molecular weight band! It looks like my Buffer EB and/or nuclease-free water are is contaminated. Have discarded both. Will re-isolated Claire and Mac’s gDNA.

Phenol-Chloroform DNA Clean Up – Mac and Claire’s Samples (from 20140410)

Due to low 260/230 values and Mac’s smeary sample, performed a phenol-chloroform DNA cleanup on the samples isolated 20140410.

  1. Brought volume of each sample to 200uL with Buffer EB (Qiagen).

  2. Added an equal volume (200uL) of 25:24:1 Phenol/Chloroform:Isoamyl alcohol.

  3. Mixed on rotator for 20mins @ RT.

  4. Separated aqueous/organic phases by spinning at 12,000g 5mins @ RT.

  5. Transferred aqueous phase to new tube. Repeated steps 2-4 until samples exhibited no more interphase. Combined aqueous phases in to a single tube for each of the two samples.

  6. Added and equal volume of chloroform (170uL).

  7. Mixed on rotator for 20mins @ RT.

  8. Separated aqueous/organic phases by spinning at 12,000g 5mins @ RT.

  9. Transferred aqueous phase to new tube.

Performed an ethanol precipitation on each sample.

  1. Added 0.1 volumes of 5M sodium acetate (pH = 5.2).

  2. Added 2 volumes of ice cold 100% EtOH.

  3. Incubated 20mins @ -20C.

  4. Pelleted DNA by spinning 16,000g, 20mins @ 4C.

  5. Discarded supe and washed pellets with 1mL 70% EtOH.

  6. Pelleted DNA by spinning 16,000g, 5mins @ 4C.

  7. Repeated steps 5-6 one time.

  8. Removed all supernatant and resuspended in 100uL of nuclease-free H2O.

  9. Spec’d on NanoDrop1000.

NOTE: Mac’s sample exhibited the same chunky/cloudiness upon addition of 100% EtOH that has been seen previously by both her and myself…


So, the clean up seemed to work wonders on the 260/230 values. Not surprisingly, Mac’s sample didn’t clean up nearly as nicely as Claire’s, based on my observations of the odd behavior during EtOH precipitation.

And, despite the nice, clean looking peaks, the 260/280 ratios are actually WORSE than the original isolation. Will run on gel for a further assessment of quality/integrity.

Loaded 5uL of each sample (~600ng) on a 1.0% agarose, 1x modified TAE gel stained with ethidium bromide.

Gel Layout:

Lane 1 – Hyperladder I (Bioline)

Lane 2 – Claire’s CgF gonad sample

Lane 3 – Mac’s gonad sample

Used Hyperladder I this time, which has a high molecular weight band of 10kb and a low molecular weight band of 200bp.

Well, this totally sucks. Both samples appear to consist of nothing but 150-200bp fragments. Is something actually degrading these samples? The Buffer EB I used during the initial extraction is certainly old. Possible source of degradation? Ugh. Maybe I’ll try this again, but resuspend in TE…

DNA Isolation – Claire’s C.gigas Female Gonad and Mac’s C.gigas Gonad

Due to the poor quality DNA yielded by the DNeasy Kit (Qiagen; see 20140404), I am re-isolating these samples using DNazol (Molecular Research Center). Weighed tissue from each frozen sample:

Claire’s (Female DNA; 5/6/2013) – 0.022g

Mac’s (EV2 9.g) – 0.017g

Incubated samples in 500uL of DNazol + 100ug/mL Proteinase K (2.7uL of 18.5mg/mL stock) O/N at RT on rotator. An additional 500uL of DNazol was added, mixed by pipetting to break up remaining tissues clumps. Manufacturer’s protocol was followed, substituting the first EtOH wash with a wash of 70% DNazol, 30% 100% EtOH. Samples were resuspended in 100uL Buffer EB (Qiagen) and spec’d on a NanoDrop1000.

NOTE: Mac’s sample seemed to get “chunky”/cloudy during the precipitation portion of the procedure. Claire’s remained clear. Although not noted, Mac’s sample behaved in a similar fashion when adding Buffer AL to the sample when using the Qiagen DNeasy Blood & Tissue Kit. Finally, Mac has previously mentioned this behavior to me as well.


Suprisingly high yields from Mac’s sample.

Both samples exhibit poor 260/230 ratios and high absorbance at 230nm is evident in both samples. Mac’s sample may benefit from

Ran ~600ng of each sample on a 0.8% 1x modified TAE agarose gel to visually assess sample quality.

Gel Loading (from left to right):

  1. Hyperladder II (Bioline)

  2. Claire’s Female DNA

  3. Mac’s gonad (EV2 9.go)

I knew the ladder was of little use due to high molecular weight of gDNA, but it still serves as a bit of a reference. Highest molecular weight band is 2000bp.

Claire’s sample looks pretty good, in relation to the lack of smearing. A single, high molecular weight band is present (albeit, faint) with almost no smearing. However, I’m disappointed by the lack of definition in the band. I fully expected a sharper, more defined band.

Mac’s sample shows a high molecular weight band and significant smearing. Smearing could be indicative of either DNA degradation or high amounts of RNA carryover. If the latter, could explain the high yield.

Will attempt to clean up both samples (RNase and/or do a chloroform clean up).

DNA gel – Claire’s C.gigas Female Gonad and Mac’s C.gigas Gonad

Ran out 2uL of Clair’es C.gigas female gonad gDNA (from 20140328) and Mac’s C.gigas gonad gDNA (from 20140402) for quality assessment. Both samples had been isolated using Qiagen’s Blood & Tissue DNeasy Kit. 2uL of each sample was run on a 0.8% 1x TBE gel.



Lane 1 – Hyperladder 1 (Bioline)

Lane 2 – Claire’s gDNA

Lane 3 – mac’s gDNA

Both samples show an extremely high amount of smearing. Additionally, both samples have definitive bands that correspond to ~1300bp and ~850bp.