DNA Isolations – Fidalgo 2SN Reciprocal Transplants Final Samplings

The remaining Olympia oysters from Jake Heare’s reciprocal transplant experiment have been retrieved from field sites and are awaiting sampling. The oysters have been stored in the cold room (temp?) for 6 days so far.

Sampling scheme is as follows:

  1. Assign unique number to oysters
  2. Photograph with ruler for future shell measurements
  3. Weigh oysters
  4. Dissect ctenidia for DNA isolation
  5. Dissect & discard viscera (e.g. digestive gland and gonad)
  6. Weigh remaining body
  7. Preserve remaining body in RNAlater
  8. Weigh empty shells


Mrunmayee photographed & initiated dissections of oysters #3 – 8. I took over for oyster #9 -14.

All oyster data is here (Google Sheet): Oly reciprocal final sampling

DNA was isolated using the E.Z.N.A. Mollusc Kit (Omega Biotek) according to the manufacturer’s protocol with the following changes:

  • No optional steps were performed
  • Ctenidia tissue was lysed for 3hrs @ 60C
  • Single elution of 50μL

Samples were stored @ -20C in: Oly gDNA Oly Reciprocal Transplant Final Sampling Box #1.

PCR – Oly RAD-seq Prep Scale PCR

Continuing with the RAD-seq library prep. Following the Meyer Lab 2bRAD protocol.

After determining the minimum number of PCR cycles to run to generate a visible, 166bp band on a gel yesterday, ran a full library “prep scale” PCR.

 

REAGENT SINGLE REACTION (μL) x11
Template 40 NA
ILL-HT1 (1μM) 5 NA
ILL-BC# (1μM) 5 NA
NanoPure H2O 5 55
dNTPs (10mM) 20 220
ILL-LIB1 (10μM) 2 22
ILL-LIB2 (10μM) 2 22
5x Q5 Reaction Buffer 20 220
Q5 DNA Polymerase 1 11
TOTAL 100 550

 

Combined the following for PCR reactions:

  • 50μL PCR master mix
  • 40μL ligation mix
  • 5μL of ILL-HT1 (1μM)
  • 5μL of ILL-BC# (1μM) – The barcode number and the respective sample are listed below.

NOTE: Samples 02, 03, & 04 did not have 40μL of the ligation reaction left (only 32μL) due to additional usage in the test scale PCR yesterday. Supplemented those three reactions with 8μL of H2O to bring them to 100μL.

 

SAMPLE BARCODE SEQUENCE
Oly RAD 02  1  CGTGAT
Oly RAD 03  2  ACATCG
Oly RAD 04  3  GCCTAA
Oly RAD 06  4  TGGTCA
Oly RAD 07  5  CACTGT
Oly RAD 08  6  ATTGGC
Oly RAD 14  7  GATCTG
Oly RAD 17  8  TCAAGT
Oly RAD 23  9  CTGATC
Oly RAD 30 10 AAGCTA

 

Cycling was performed on a PTC-200 (MJ Research) with a heated lid:

STEP TEMP (C) TIME (s)
Initial Denaturation
  • 98
  • 30
12 cycles
  • 98
  • 60
  • 72
  • 5
  • 20
  • 10

 

After cycling, added 16μL of 6x loading dye to each sample.

Due to limitations in available comb sizes and inability to combine combs to make larger well sizes, only loaded 58μL of samples in each well on this gel. Will load remainder on a second gel and combine after PCR products are purified.

Results:

 

Well, this is lame. There are absolutely no PCR products on this gel. In fact, this just looks like big smears of degraded DNA. I was expecting an amplicon of ~166bp to cut out of the gel. Based off of the test scale PCR from yesterday, everything should have been hunky dory. Not really sure what to think about this…

PCR – Oly RAD-seq Test-scale PCR

Continuing with the RAD-seq library prep. Following the Meyer Lab 2bRAD protocol.

Prior to generating full-blown libraries, we needed to run a “test-scale” PCR to identify the minimum number of cycles needed to produce the intended product size (166bp).

I ran PCR reactions on a subset (Sample #: 2, 3, & 4) of the 10 samples that I performed adaptor ligations on Friday.

PCR reactions were set up on ice in 0.5mL PCR tubes.

REAGENT SINGLE REACTION (μL) x4.4
Template 8 NA
NanoPure H2O 1 4.4
dNTPs (1mM) 4 17.6
ILL-LIB1 (10μM) 0.4 1.76
ILL-LIB2 (10μM) 0.4 1.76
ILL-HT1 (1μM) 1 4.4
ILL-BC1 (1μM) 1 4.4
5x Q5 Reaction Buffer 4 17.6
Q5 DNA Polymerase 0.2 0.88
TOTAL 20 52.8

 

Combined 12μL of master mix with 8μL of the ligation reaction from earlier today.

Cycling was performed on a PTC-200 (MJ Research) with a heated lid:

STEP TEMP (C) TIME (s)
Initial Denaturation
  • 98
  • 30
27 cycles
  • 98
  • 60
  • 72
  • 5
  • 20
  • 10

We’re following the “1/4 reduced representation” aspect of the protocol. As such, 5μL of each reaction was pulled immediately after the extension (72C – machine was paused) of cycles 12, 17, 22, & 27 in order to determine the ideal number of cycles to use. Also ran the ligation reactions (labeled “Ligations” on the gel below) of the samples as a pre-PCR comparison. Treated them the same as the PCR reactions: mixed 8μL of the ligation with 12μL of H2O, used 5μL of that mix to load on gel.

These samples were run on a 1x modified TAE 2% agarose gel (w/EtBr).

 

Results:

 

 

Test-scale PCR gel. Green arrow indicates desired band. The numbers below the headings indicate the sample number.

 

 

This looks pretty good. The green arrow on the gel indicates the desired band size (~166bp). Although difficult to see on this gel image, there is a gradient in band intensities across the cycles (band intensity increases as cycle number increases). Looks like we can use 12 cycles for our PCRs.

One other aspect of this gel that is very interesting is the ligations. The three ligation samples all show an intact high molecular weight band! This is very surprising, since the input gDNA from these three samples does not look this.

Adaptor Ligation – Oly AlfI-Digested gDNA for RAD-seq

Continued to follow the 2bRAD protocol (PDF) developed by Eli Meyer’s lab.

Digested DNA from earlier today was not run out on a gel due to the fact that the input gDNA was degraded and a shift in the high molecular weight band (indicating the digestion was successful) would not exist because a high molecular weight band is absent in these samples.

 

Anneal Adaptors

After preparing the two adaptors below, they were incubated for 10mins @ RT:

  • Adaptor 1 (2μM final concentration of each oligo): 1.5μL of 5ILL-NR (100μM) + 1.5μL of anti-ILL (100μM) + 72μL H2O = 75μL total
  • Adaptor 2 (2μM final concentration of each oligo): 1.5μL of 3ILL-NR (100μM) + 1.5μL of anti-ILL (100μM) + 72μL H2O = 75μL total

After annealing, the adaptors were stored on ice.

 

Adaptor Ligation

All components were stored on ice. Ligation reactions were prepared on ice and performed in 0.5mL snap cap tubes.

REAGENT SINGLE REACTION (μL) x11
Digested DNA 10 NA
ATP (10mM) 1 11
10x T4 Ligase Buffer 4 44
Adaptor 1 (2μM) 5 55
Adaptor 2 (2μM) 5 55
T4 DNA Ligase 1 11
NanoPure H2O 24 264
TOTAL 50 440

Combined 40μL of the master mix with 10μL of AlfI-digested DNA in a 0.5mL snap cap tube.

Incubated ligation reaction @ 16C for 3hrs in PTC-200 thermal cycler (MJ Research) – no heated lid.

Ligations were stored @ -20C until I can continue working with them on Monday.

Restriction Digest – Oly gDNA for RAD-seq w/AlfI

Previously initiated the RAD-seq procedure for the sample set described below. However, the test scale PCR yielded poor results. Katherine Silliman suggested that the poor performance of the test scale PCR was likely due to low numbers of adaptor-ligated fragments. Since the input DNA is so degraded, I’ve repeated this using 9μg of input DNA (instead of the recommended 1.2μg). This should increase the number of available cleavage sites for AlfI, thus improving the number of available ligation sites for the adaptors.

Used a subset (10 samples) from the Ostrea lurida gDNA isolated 20150916 to prepare RAD libraries.

Followed the 2bRAD protocol (PDF) developed by Eli Meyer’s lab.

Prepared 9.0μg of each of the following samples in a volume of 10μL:

Google Sheet: 20151009_RADseq_DNA_calcs

 

Prepared a 150μM working stock of the SAM buffer needed for the restriction digestion by diluting 30μL of the supplied stock (500μM) in 70μL NanoPure H2O (total volume = 100μL). This working stock was stored @ -20C in FTR 209 in the “RAD-seq Reagents” box.

Prepared master mix for restriction enzyme reaction:

REAGENT SINGLE REACTION (μL) x11
DNA 8 NA
10x Buffer R 1.2μL 13.2μL
150μM SAM 0.8μL 8.8μL
AlfI 0.5μL 5.5μL
H2O 1.5μL 16.5μL

 

Combined 4μL of the master mix with 8μL of each sample in 0.5mL snap cap tubes. Incubated @ 37C 2hrs. in thermal cycler (PTC-200; no heated lid). Heat inactivated the digest @ 65C for 10mins.

Sample Submission – Additional Geoduck gDNA for Genome Sequencing @ BGI

Previous shipment of gDNA proved to be of insufficient quantity when assessed by BGI, so needed to isolate more.

Shipped the pooled gDNA we’ve been accumulating to BGI to contine the geoduck genome sequencing project.

Sample was shipped on dry ice with the appropriate paperwork required by BGI (sample declaration letter).

Assigned BGI Lot: 1510071003

Sample Submission – Additional Olympia Oyster gDNA for Genome Sequencing @ BGI

Previous shipment of gDNA proved to be of insufficient quantity when assessed by BGI, so needed to isolate more.

Shipped the pooled gDNA we’ve been accumulating to BGI to contine the geoduck genome sequencing project.

Sample was shipped on dry ice with the appropriate paperwork required by BGI (sample declaration letter).

Assigned BGI Lot: 1510071002

Agarose Gel – Geoduck & Olympia Oyster gDNA

Needed to assess the integrity of the newest gDNA isolated for the two genome sequencing projects: Geoduck gDNA from earlier today and Olympia oyster gDNA from 20151002.

Also needed to assess the integrity of the gDNA of ethanol-preserved Olympia oyster mantle tissue from Jake’s reciprocal transplant experiment, isolated on 20151002: samples NF1A & SN49A.

Ran samples on a 0.8% agarose, 1X modified TAE gel.

Loaded 1μL (~300ng) of the geoduck, Oly and NF1A samples.

Loaded 2μL (~100ng) of the SN49A sample.

Used 5μL of ladder.

Results:

 

Genome Sequencing Samples

The geoduck and the Oly samples look good. Intact, high molecular weight band. My only concern is the noticeable difference in band intensities between these two. Both samples should be ~300ng/μL, based on the NanoDrop1000 readings. However, it’s evident that the concentrations of these two samples differ greatly. Additionally, we can use the ladder to gauge the concentrations of the samples, since I loaded 0.5μg of the ladder, which is the quantity referenced on the ladder guide above.

It would appear that the geoduck sample concentration is closer to 60ng/μL (band intensity is similar to that of the 500, 1000, & 3000bp markers), as opposed to the 292ng/μL that the NanoDrop1000 indicated.

The Oly sample appears to have even less and appears less intense than the lowest concentration bands on the marker (16.0ng/μL). That’s not even remotely close to the 331ng/μL measured by the NanoDrop1000.

It’s difficult to say why this might be, as both samples were RNased and neither of them show extensive smearing (both of those factors would contribute to inflated spec readings).

Regardless, will ship them off to BGI to supplement the previous gDNA for this project.

Ethanol-Preserved Samples

Both samples show extensive smearing and no high molecular weight band, indicating they are both completely degraded. This is a very bad result for this project, as the tissue in this group is/was a bit of grasping at straws to obtain some intact DNA to use for the RAD-seq that we intend to pursue.

DNA Quantification – Pooled geoduck gDNA

Pooled the gDNA samples from earlier today & from 20151002.

The pooled volume = 260μL

Quantified on the Roberts Lab NanoDrop1000.

Results:

 

Besides the weird peak at 240nm, everything looks great – perfect 260/280 & 260/230 ratios.

Yield = 75.92μg

This gDNA has been RNase treated, so the concentration should be semi-accurate. Regardless, even if there’s a discrepancy of 50%, this should provide enough additional DNA for BGI to work with.

Will run on gel to evaluate integrity and then send to BGI.

gDNA Isolation – Geoduck Adductor Muscle

My isolation on Friday didn’t yield a sufficient quantity of gDNA for the additional DNA needed for the geoduck genome sequencing project. Used two adductor muscles (Box 1) samples collected by Brent & Steven on 20150811.

Tissue weights:

  • Geoduck adductor 1: 433mg (gone)
  • Geoduck adductor 2: 457.4mg (gone)

 

Samples were isolated using DNAzol (Molecular Research Center) according to the manufacturer’s protocol, with the following adjustments:

 

  • Tissues homogenized in 750μL of DNAzol with disposable mortar/pestle tubes using 10 pestle strokes
  • After homogenization, topped off tubes to 960μL with DNAzol, added 40μL RNAse A (100mg/mL) and incubated @ RT for 15mins.
  • Performed optional centrifugation step (10,000g, 10mins @ RT)
  • Initial pellet wash was performed using a 70%/30% DNAzol/EtOH
  • Pellets were resuspended Buffer EB (Qiagen)
  • Insoluble material was pelleted (12,000g, 10mins @ RT) and supe transferred to new tubes

NOTE: Both samples produced a stark white, “cottony” precipitate after the addition of the ethanol. This precipitate was transferred to a clean tube and processed in the same fashion.

 

Resuspension volumes

Adductor 1:  200μL

Adductor 2: 50μL

Adductor 1 & 2 fluff: 500μL each

 

Spec’d on Roberts Lab NanoDrop1000.

Results:

 

NOTE: The sample labeled “gDNA geoduck adductor 1″ is actually adductor 2. The sample labeled “gDNA geoduck adductor 1{1} is actually adductor 1. However, this is probably moot since these two samples will be pooled shortly.

I’m not going to speculate why there’re weird peaks at 240nm…

The two “fluff” samples aren’t good (extremely high 260/280 ratios, very low 260/230 ratios, and weird peak at 240nm). Not sure what the fluff is that precipitated out with the EtOH addition. Will discard them.

The two normal samples look fine. Will use them for pooling.

Yields

Adductor 1: 52.2μg

Adductor 2: 8.25μg