Tag Archives: bioanalyzer

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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Library Cleanup – LSU C.virginica MBD BS Library

I was contacted by the sequencing facility at the University of Oregon regarding a sample quality issue with our library.  As evidenced by the electropherogram below, there is a great deal of adaptor primer dimer (the peak at 128bp):

 

This is a problem because such a high quantity of adaptor sequence will result in the majority of reads coming off the Illumina being just adaptor sequences.

With the remainder of the library sample prepared earlier, I performed the recommended clean up procedure for removing adaptor sequences in the EpiNext Post-Bisulfite DNA Library Preparation Kit – Illumina (Epigentek).    Briefly:

  • Brought sample volume up to 20uL with NanoPure H2O (added 9.99uL)

  • Added equal volume of MQ Beads

  • Washed beads 3x w/80% EtOH

  • Eluted DNA w/12uL Buffer EB (Qiagen)

After clean up, quantified the sample via fluorescence using the Quant-iT DNA BR Kit (Life Technologies/Invitrogen).  Used 1uL of the sample and the standards.  All standards were run in duplicate and read on a FLx800 plate reader (BioTek).

Results are here: 20150122 – LSU_virginicaMBDlibraryCleanup

Library concentration = 2.46ng/uL

Brought the entire sample up to 20uL with Buffer EB (Qiagen) and a final concentration of 0.1% Tween-20 (required by the sequencing facility).

Sent sample to the University of Oregon to replace our previous submission.

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Bioanalyzer – Fragmented SB/WB gDNA (from 20100625)

To gain a more quantitative assessment of the fragmentation from 20100625, I ran 1uL of each sample (~55ng, according to pre-fragmentation spec values) on the Agilent Bioanalyzer 2100, using the DNA 1000 kit, according to manufacturer’s protocol.

Results:

Avg. size of fragmentation is ~460bp for the two samples. Fragmentation size was determined by marking the same region on both sample’s electropherograms (see below).

R37: Avg. size = 450bp (in Region 1, marked with blue lines in image below)

 

 

R51: Avg. size = 468bp (in Region 1, marked with blue lines in image below)

 

 

 

Overlay of R37 and R51 fragmentation. Note that both electropherograms are nearly identical (this is good).

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cDNA clean up & Bioanalyzer for SOLiD Libraries – Abalone, Yellow Perch, Lake Trout, Herring

Amplified cDNA was cleaned up using the Invitrogen PureLink Micro Kit, but was done so according to Ambion’s Whole Transcriptome Analysis Kit protocol and then spec’d.

Results:

0.5uL was removed from each sample and mixed with 0.5uL to run on DNA 1000 chips on the Bioanalyzer 2100. The slideshow below shows the electropherograms from each sample. Each sample (to be considered worthy of moving to the next stage) should have <20% of the sample in the 25-150bp range. All 8 samples exhibit this and their peaks look very good. Will proceed to ePCR/templated bead prep next week.

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Bioanalyzer Total, mRNA and post-fragmentation SOLiD Libraries – Abalone pools

0.5uL of fragmented mRNA from each library (combined with 0.5uL) was run on Agilent Bioanalyzer 2100 using RNA Pico chips/reagents according to Agilent’s protocol.

Results:

Total RNA shows a single, distinct rRNA band, along with some low-molecular weight RNA (i.e. degraded) in both total RNA samples. mRNA samples exhibit the expected “smear” that spans a large range of molecular weights. Both mRNA samples also show residual rRNA bands, but their concentrations should be extremely low. Fragmented samples show the expected strong band of low-molecular weight RNA. The CE frag sample exhibits some larger banding, which is probably background signal (compare to the empty lane labelled “Sample 7″).

Will proceed with rest of library procedure for both fragmented samples.

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Bioanalyzer for SOLiD Libraries – Fragmented mRNA from Perch, Lake Trout & Herring RNA samples

1uL of each sample from 20100325 was run on the Agilent 2100 Bioanalyzer on a RNA Pico 6000 chip to evaluate RNA quantity and fragmentation.

Results:

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Bioanalyzer for SOLiD libraries – Total and mRNA from Perch, Lake Trout & Herring RNA samples (CONTINUED from yesterday)

Total and mRNA aliquots (~5ng/uL) were run on the Agilent Bioanalyzer Pico RNA chips.

Results:

The gel below shows the comparison/results of total RNA and subsequent mRNA isolations. The gel indicates the following:

  1. The HPWS09 total RNA (Herring) is totally degraded, but shows the expected profile in the mRNA prep. It would be extremely interesting to see if the degradation has any effect on sequencing, as the mRNA will get fragmented any way in the next step of library construction.

  2. mRNA isolations worked for all samples. Although one might be inclined to say that mRNA isolation did NOT work for the WB sample, one has to take in to consideration that the gel software adjusts the gel contrast to enhance low signals. That’s why all the mRNA samples exhibit a dark background. mRNA generates a broad, relatively weak signal when compared to a total RNA sample. So, the software attempts to boost the low signal for display purposes. Thus, if we were to decrease this signal boosting (or contrast) for the WB mRNA so that the background color matched the WB total RNA background color (white), the rRNA bands visible in the WB mRNA sample would fade to a point where they would not be visible. See the electropherogram overlay (below the gel) for a more visual comparison of this concept.

Electropherogram Overlays of WB total RNA and WB rRNA

The WB total RNA is the red graph which shows extremely high levels of rRNA (as expected). After subsequent mRNA isolation (the blue graph), the rRNA is virtually gone and no longer comprises a significant portion of the sample.

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Bioanalyzer – Herring Liver cDNA for SOLiD Libraries

Samples were run on the DNA 1000 chip for cDNA smear analysis.

Results: 2 of the 4 samples (2L & 3L) look perfect (<20% of cDNA in the 25-150bp range). 6L has <20% of the cDNA in the 25-150bp range (which is perfect), but exhibits a “smear” from ~250-500bp. cDNA in this range suggests overamplification, which will skew gene expression profile. Can repeat PCR for 6L using outer gel slices and reduce the number of cycles to prevent overamplification, if desired. Spoke to Steven and since these samples won’t be used to evaluate gene expression (they’re for SNP discovery), we won’t worry about it for the time being.

Sample 4L has some very strange signals being generated in the ~500-800bp range. Additionally, the virtual gel image (not shown) shows a great deal of smearing, unlike the other samples.

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RNA Fragmentation – Herring Liver mRNA for SOLiD Libraries

Samples from 20091203. 0.5uL was removed from each and transferred to separate tubes and diluted to < 5ng/uL for subsequent Bioanalyzer analysis using the Pico chip. Samples were fragmented using RNase III according to the Ambion WTK protocol and then cleaned up/concentrated using the Invitrogen RiboMinus Concentration Module according to the Ambion WTK protocol.

Samples were spec’d prior to running on the Bioanalyzer:

Concentrations/absorbance values are not accurate when using the NanaDrop after using the RiboMinus Concentration module, according to the Ambion WTK protocol. However, yields seem pretty good…

Total, mRNA and fragmented mRNA from each of the four samples was run on the Pico chip with the Eukaryote Total RNA Bioanalyzer protocol.

Results:

The 2L tot (total RNA) and 3L tot (total RNA) samples are clearly very good quality. 2L tot does exhibit some very slight degradation, though. 4L tot (total RNA) and 6L tot (total RNA) show a much greater degree of degradation. All mRNA samples show complete removal of any trace, contaminating rRNA. The fragmented samples (the last four samples on the gel image above) all appear to be perfect. The 4L frag sample simply has less RNA loaded and that is why it is not as dark as the other three fragmented samples. Despite the degradation in the 4L tot and 6L tot samples, the fragmentation profile looks good and we will proceed with making the cDNA libraries for those samples.

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Bioanalyzer Submission – Trout RBC, Colleen’s gigas GE sample, Mac’s DH/BB PCR for SOLiD WTK

Samples were delivered for analysis on the DNA 1000 chip.

UPDATE 20091008 They do not have the DNA 1000 kit in stock. Will be using High Sensitivity Kit instead. Will have in stock on Tuesday.

Results:

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