DNA Isolation – Geoduck Adductor Muscle gDNA

Since we still don’t have sufficient gDNA for the full scope of the genome sequencing, I isolated more gDNA.

Isolated gDNA from 257mg adductor muscle tissue collected by Steven & Brent on 20150811.

Tissue was thoroughly minced with a clean razor blade and then processed with the E.Z.N.A. Mollusc Kit (Omega BioTek) with the following changes:

  • Doubled solution volumes for steps before sample was loaded on columns
  • Sample was split equally in two tubes prior to addition of 100% EtOH
  • All mixing was done by shaking – no vortexing! Done this way to, hopefully, maintain gDNA integrity
  • Elution volume = 50μL
  • Elution was repeated using the initial elution to maximize recovery while maintaining low sample volume.
  • The two preps were pooled – final volume = 79μL

DNA was quantified using two methods: NanoDrop1000 & QuantIT dsDNA BR Kit

For the Quant-IT kit, the samples were quantified using the QuantIT dsDNA BR Kit (Invitrogen) according to the manufacturer’s protocol.

Standards were run in triplicate, samples were run in duplicate.

96-well black (opaque) plate was used.

Fluorescence was measured on the Seeb Lab’s Victor 1420 plate reader (Perkin Elmer).

Results:

METHOD CONCENTRATION (ng/μL) VOLUME (μL) YIELD (ng)
NanoDrop1000 54.93 79 4,339
Quant-IT 34.52 79 2,727

 

The NanoDrop1000 overestimates the concentration of the sample by 1.6x!

Regardless, the yield isn’t all that great, which has generally been the case for all of the geoduck gDNA isolations I’ve performed. It would probably be prudent to try isolating gDNA from a different tissue to see if yields improve…

Will evaluate gDNA quality on a gel.

Fluorescence (Google Sheet): 20151124_geoduck_oly_gDNA_quants

 

NanoDrop1000 Measurements and Plots

DNA Isolation – Olympia Oyster Outer Mantle gDNA

Since we still don’t have sufficient gDNA for the full scope of the Olympia oyster genome sequencing, I isolated more gDNA.

Isolated gDNA from 118mg outer mantle tissue collected by Steven & Brent on 20150812.

Tissue was thoroughly minced with a clean razor blade and then processed with the E.Z.N.A. Mollusc Kit (Omega BioTek) with the following changes:

  • Doubled solution volumes for steps before sample was loaded on columns
  • Sample was split equally in two tubes prior to addition of 100% EtOH
  • All mixing was done by shaking – no vortexing! Done this way to, hopefully, maintain gDNA integrity
  • Elution volume = 50μL
  • Elution was repeated using the initial elution to maximize recovery while maintaining low sample volume.
  • The two preps were pooled – final volume = 79μL

DNA was quantified using two methods: NanoDrop1000 & QuantIT dsDNA BR Kit

For the Quant-IT kit, the samples were quantified using the QuantIT dsDNA BR Kit (Invitrogen) according to the manufacturer’s protocol.

Standards were run in triplicate, samples were run in duplicate.

96-well black (opaque) plate was used.

Fluorescence was measured on the Seeb Lab’s Victor 1420 plate reader (Perkin Elmer).

Results:

METHOD CONCENTRATION (ng/μL) VOLUME (μL) YIELD (ng)
NanoDrop1000 552.53 79 43,650
Quant-IT 219.07 79 17,307

 

The NanoDrop1000 overestimates the concentration of the sample by 2.5x!

Regardless, this is a solid yield and, when combined with the other Ostrea lurida gDNA that I cleaned up today, should push the total amount of gDNA submitted to BGI over the required threshold.

Will evaluate gDNA quality on a gel.

Fluorescence (Google Sheet): 20151124_geoduck_oly_gDNA_quants

 

NanoDrop1000 Measurements and Plots

 

DNA Quantification – MBD-enriched Olympia oyster DNA

Quantified the MBD enriched samples prepped over the last two days: MBD enrichment, EtOH precipiation.

Samples were quantified using the QuantIT dsDNA BR Kit (Invitrogen) according to the manufacturer’s protocol.

Standards were run in triplicate, samples were run in duplicate.

96-well black (opaque) plate was used.

Fluorescence was measured on the Seeb Lab’s Victor 1420 plate reader (Perkin Elmer).

Results:

Google Sheet: 20151123_MBD_libraries_quantification

Standard curve looked good – R² = 0.999

MBD recovery ranged from ~250 – 600ng.

MBD percent recoveries ranged from ~2 – 20%. Input DNA quantities were taken from Katherine’s numbers (Google Sheet): Silliman-DNA-Samples

Will contact services about getting bisulfite Illumina sequencing performed.

Ethanol Precipitation – Olympia oyster MBD

Precipitated the MBD enriched DNA from yesterday according to the MethylMiner Methylated DNA Enrichment Kit (Invitrogen) protocol.

However, since the protocol has two elution steps that are each saved separately from each other for each sample, I did the following to combine the two elution fractions into a single sample:

  • Pelleted one elution fraction from each sample
  • Discarded supernatant from pelleted sample
  • Transferred second elution fraction to the pellet from the first elution fraction
  • Pelleted second elution fraction

The rest of the ethanol precipitation procedure was followed per the manufacturer’s protocol.

Final pellets were resuspended in 25μL of Buffer EB (Qiagen) and stored @ 4C.

MBD enriched DNA will be quantified tomorrow.

MBD Enrichment – Sonicated Olympia Oyster gDNA

Olympia oyster gDNA that had previously been sonicated and fragmented was enriched for the methylated fragments using the MethylMiner Methylated DNA Enrichment Kit (Invitrogen).

Prepared the following components:

  • 20mL 1x Bind/Wash Buffer (4mL 5x Bind/Wash Buffer + 16mL H2O)
  • 640μL of beads (35μL of beads x 18 samples )
  • 200μL MBD-Biotin Protein (63μL MBD-Biotin Protein + 137μL 1x Bind/Wash Buffer)

Followed the manufacturer’s protocol for input DNA quantities 1μg – 10μg.

Used single fraction, high salt elution.

Neglected to account for the control reaction during initial set up and did not have sufficient quantities of beads to run a control reaction.

The table below provides the individual sample volumes and the volumes of the buffer, beads, H2O for the MBD capture reactions.

Samples listed with “NA” were not processed because they did not fragment during sonication.

Sample Volume (μL) Buffer/Beads (μL) H2O (μL) Total (μL)
hc1_2B 75 135 290 500
hc1_4B 90 135 275 500
hc2_15B 75 135 290 500
hc2_17 75 135 290 500
hc3_1 75 135 290 500
hc3_5 75 135 290 500
hc3_7 70 135 295 500
hc3_9 NA NA NA NA
hc3_10 70 135 295 500
hc3_11 70 135 295 500
ss2_9B 190 135 175 500
ss2_14B 195 135 170 500
ss2_18B 195 135 170 500
ss3_3B 190 135 175 500
ss3_4B NA NA NA NA
ss3_14B 195 135 170 500
ss3_15B 195 135 170 500
ss3_16B 195 135 170 500
ss3_20 135 135 230 500
ss5_18 75 135 290 500

 

Non-captured & wash fractions were pooled into single samples and stored @ -20C.

MBD fraction was EtOH precipitated according to the manufacturer’s protocol and incubate O/N @ -80C.

 

DNA Sonication – Oly gDNA for MBD

In preparation for MBD enrichment, fragmented Olympia oyster gDNA with a target size of ~350bp.

Genomic DNA samples were isolated and provided to us by Katherine Silliman at UIC. Selected samples will compare Hood Canal (HC) and Oyster Bay (SS, South Sound) populations.

Used the Seeb Lab’s Bioruptor 300 (Diagenode) sonicator.

After sonication, samples were run on a the Seeb Lab’s 2100 Bioanalyzer (Agilent) on DNA 12000 chips.

Results:

HOOD CANAL SAMPLES

 

OYSTER BAY SAMPLES

More detailed analysis (including average fragment size for each samples) will be coming soon…

qPCR – Oly RAD-Seq Library Quantification

After yesterday’s attempt at quantification revealed insufficient dilution of the libraries, I repeated the qPCRs using 1:100000 dilutions of each of the libraries. Used the KAPA Illumina Quantification Kit (KAPA Biosystems) according to the manufacturer’s protocol.

Made 1:100000 dilutions of each library were made with NanoPure H2O.

Ran all samples, including standards, in triplicate on the Roberts Lab Opticon2 (BioRad).

Plate set up and master mix can be found here: 20151117_qPCR_plate_layout_Oly_RAD.JPG

 

Results:

qPCR Data File (Opticon2): Sam_20151117_100745.tad

qPCR Data (Google Sheet): 20151117_RAD_qPCR_data

Overall, the new dilutions worked well, with all the library samples coming up between Ct 9 – 15, which is well within the range of the standard curve.

Manually adjusted the baseline threshold to be above any background fluorescence (see images below).

All samples, except Oly RAD 30, exhibit two peaks in the melt curve indicating contaminating primer dimers. Additionally, the peak heights appear to be roughly equivalent. Can we use this fact to effectively “halve” the concentration of our sample to make a rough estimate of library-only PCR products?

 

Here are the calculated library concentrations, based on the KAPA Biosystems formulas

Library Library Stock Conc. (nM) Stock Halved (nM)
Oly RAD 02 46.70 23.35
Oly RAD 03 79.35 39.67
Oly RAD 04 61.35 30.67
Oly RAD 06 30.61 15.30
Oly RAD 07 477.05 238.53
Oly RAD 08 46.32 23.16
Oly RAD 14 224.91 112.46
Oly RAD 17 24.56 12.28
Oly RAD 23 49.56 24.78
Oly RAD 30 11.19  NA

 

Amplification plots of standard curve samples:

 

 

Melt curve plots of standard curve samples. Shows expected “shoulder” to the left of the primary peak:

 

 

 

Amplification plots of RAD library samples:

 

 

Melt curve plots of RAD library samples. Peak on the right corresponds to primer dimer. Peak heights between primer dimer and desired PCR product are nearly equivalent for each respective sample, suggesting that each product is contributing equally to the fluorescence generated in the reactions:

 

 

Melt curve plot of Oly RAD library 30. Notice there’s only a single peak due to the lack of primer dimers in this sample:

qPCR – Oly RAD-Seq Library Quantification

The final step before sequencing these 2bRAD libraries is to quantify them. Used the KAPA Illumina Quantification Kit (KAPA Biosystems) according to the manufacturer’s protocol.

Made 1:4 dilutions of each library to use as template.

Ran all samples, including standards, in triplicate on the Roberts Lab Opticon2 (BioRad).

Plate set up and master mix can be found here: 20151116_qPCR_plate_layout_Oly_RAD.JPG

 

Results:

qPCR Data File (TAD): Sam_20151116_144718.tad

The take home messages from this qPCR are this:

  • The amplification plots that are pushed up against the left side of the graph (essentially at ~ cycle 1) are all of the libraries. A 1:4 dilution was insufficient to have the libraries amplify within the range of the standard curve.
  • All libraries except one (Oly RAD Library 30) have detectable levels of primer dimer. This confounds library quantification (because both the intended PCR product and the primer dimers contribute to the fluorescence accumulation), as well as potentially interfering with the subsequent Illumina sequencing (primer dimers will be sequenced and contain no insert sequence).

Will repeat the qPCR with more appropriately diluted libraries.

See the info below for more deets on this run.

 

 

Default analysis settings need to be adjusted to account for how early the standard curve comes up. Otherwise, the Opticon software sets the baseline incorrectly:

 

 

 

The KAPA Quantification Kit indicates that the baseline calculations need to be extended to cycles 1 through 3. This allows the software to set the baseline threshold correctly:

 

 

 

Melt curve analysis of the standard curve shows the expected profile – slight hump leading into the peak:

 

 

 

Melt curve analysis of the libraries. Dual peaks indicate primer dimer contamination:

 

 

Melt curve analysis of Oly RAD Library 30. Shows the desired single peak, suggesting library is free of primer dimers:

Gel Extraction – Oly RAD-Seq Prep Scale PCR

Extracted the PCR products from the gel slices from 20151113 using the QIAQuick Gel Extraction Kit (Qiagen) according to the manufacturer’s protocol. Substituted MiniElute columns so that I could elute with a smaller volume than what is used in the QIAQuick standard protocol.

Samples were eluted with 20μL of Buffer EB.

Will quantify these libraries via qPCR.

DNA Quality Assessment – Geoduck, Oly & Oly 2SN

I recently ran gDNA isolated for geoduck and Olympia oyster genome sequencing, as well as gDNA isolated from the Olympia oyster reciprocal transplant experiment out on a Bioanalyzer (Agilent) using the DNA 12000 chips. The results from the chip were a bit confusing and difficult to assess exactly what was going on with the DNA.

So, I ran 5μL of each of those samples on a 0.8% agarose 1x modified TAE gel w/EtBr to get a better look at how the samples actually looked.

Results:

 

Both the geoduck and the Olympia oyster samples for genome sequencing show intact, high molecular weight bands with some smearing (i.e. degradation). The oly sample looks a bit funky, most likely due to a gel anomaly. I’ll quantify these using a dye-based method for a more accurate quantification before sending off to BGI.

The Fidalgo 2SN samples all have intact, high molecular weight bands, but most of the samples show extensive smearing (i.e. degradation). However, sample 2SN 35 has no visible DNA at all.

Here’s a table highlighting the differences between the Fidalgo gDNA samples:

Sample Fresh/Frozen Isolator
10 Fresh Sam
11 Fresh Sam
12 Fresh Sam
20 Fresh Mrunmayee
21 Fresh Mrunmayee
22 Fresh Mrunmayee
32 Frozen Sam
33 Frozen Sam
34 Frozen Sam
35 Frozen Sam

 

The fresh ctenidia samples were isolated by me on 20151021 and by Mrunmayee on 20151023. The frozen ctenidia samples were isolated by me on 20151103.

It’s interesting to note that Mrunmayee’s isolations appear to exhibit the least amount of degradation. Besides her handling the samples, the primary difference is that her samples were incubated in the buffer/Pro K solution O/N @ 37C, while my fresh samples were incubated @ 60C for 3hrs and my frozen samples were incubated @ 60C for 1hr. Overall, though, the frozen samples look the worst.

Finally, it’s also interesting to see that the two samples isolated using DNazol (geoduck and Olympia oyster genome samples) migrate more slowly than the remaining Olympia oyster samples which were isolated with the E.Z.N.A. Mollusc Kit.