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 55
ILL-BC# (1μM) 5 NA
NanoPure H2O 5 55
dNTPs (1mM) 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:

  • 55μL PCR master mix
  • 40μL ligation mix
  • 5μL of ILL-BC# (1μM) – The barcode number and the respective sample are listed below.

 

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
17 cycles
  • 98
  • 60
  • 72
  • 5
  • 20
  • 10

 

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

Loaded 10μL of ladder on each of the two gels.

Results:

 

Things looked fine. Excised the bands from each sample indicated by the green arrow. Before and after gel images show regions excised. Will purify the bands and quantify library yields.

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, 17, & 30) of the 10 samples that I performed adaptor ligations on 20151029.

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 1.2% agarose gel (w/EtBr).

 

Results:

Gel image denoting sample numbers within each cycle number. Green arrow indicates the expected migration of our target band size of 166bp.

Looks like cycle 17 is the minimum cycle number with which we begin to see a consistent ~166bp band. Will continue on with the “prep-scale” PCR using 17 cycles.

DNA Quantification & Quality Assessment – Oly 2SN gDNA

Comparison of three different approaches to using the E.Z.N.A. Mollusc Kit:

  • Fresh isolations by me
  • Fresh isolations by Mrunmayee
  • Isolations from tissue frozen in buffer by me

Results:

Bioanalyzer Data File (XAD file): 2100 expert_DNA 12000_DE72902486_2015-11-04_15-06-32.xad

There’s a LOT going on here. Will update this entry tomorrow with more info.

DNA Quantification & Quality Assessment – Geoduck & Oly gDNA

Quantified the following samples with the Roberts Lab NanoDrop1000 (ThermoFisher) and assessed DNA integrity on the Seeb Lab Bioanalyzer 2100 (Agilent) using the DNA 12000 chip assay:

Results:

Bioanalyzer Data File (XAD file): 2100 expert_DNA 12000_DE72902486_2015-11-04_15-06-32.xad

 

OK, there’s a LOT going on here. Will update this notebook with my thoughts sometime tomorrow…

 

DNA Isolations – Oly Fidalgo 2SN Ctenidia

Isolated DNA from 24 2SN ctenidia samples from Friday’s sampling (#32 – 55). Samples were thawed at RT.

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

  • Samples were incubated @ 60C for only 1hr, per Steven’s recommendation (an attempt to prevent degradation)
  • No optional steps were performed
  • Used 300μL of MBL Buffer for all samples (this was more than the recovered volume of aqueous phase from each sample)
  • Single elution of 50μL

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

Some notes:

  • Total time (including 1hr incubation): 4.5hrs.
  • Short incubation time did not completely digest samples
  • Partial tissue digestions led to difficulties in recovering entire aqueous phase, post chloroform treatment

 

Oyster Sampling – Oly Fidalgo 2SN, 2HL, 2NF 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 15 days so far.

The previous sampling scheme was described here: DNA Isolations – Fidalgo 2SN Reciprocal Transplants Final Samplings

Sampling scheme for today was as follows:

  1. Assign unique number to oysters (1-100 for each of the three populations)
  2. Photograph with ruler for future shell measurements
  3. Weigh oysters
  4. Dissect ctenidia for DNA isolation in 350μL MBL1 Buffer + 25μL Proteinase K (reagents part of the E.Z.N.A. Mollusc Kit [Omega BioTek)
  5. Preserve portion of remaining body tissue (not viscera; gonad/digestive gland) in 1mL RNAlater (Life Technologies)

Ctenidia samples were stored -80C in the buffer/pro k solution for DNA isolation at a later date.

RNAlater samples will be stored over the weekend at 4C and then transferred to -20C for long term storage.

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

All photos from today’s sampling are here: Oyster Measurement Photos

DNA Isolation – Geoduck & Olympia Oyster

Amazingly, we need more gDNA for the two genome sequencing projects (geoduck and Olympia oyster). Used geoduck adductor muscle sample from Box 1 of the geoduck samples collected by Brent & Steven on 20150811. Used Olympia oyster ctenidia from Box 1 of adductor muscle sample collected by Brent & Steven on 20150812.

Tissues were split in approximately half, minced and transferred to tubes with 1mL of DNAzol + 50μg/mL of Proteinase K (Fermentas). Previously, I had just homogenized samples. I’m hoping that the overnight digestion with Proteinase K will help increase yields from these.

Tissue weights:

  • Geoduck adductor muscle tube 1: 292mg (gone)
  • Geoduck adductor muscle tube 2: 320mg (gone)
  • Olympia oyster ctenidia tube 1: 135mg (gone)
  • Olympia oyster ctenidia tube 2: 130mg (gone)

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

 

  • Samples were incubated O/N @ RT on a rotator.
  • After Proteinase K digestion, 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)

Resuspension volume = 500μL total for each species

Samples were incubated O/N at RT to facilitate pellet resuspension.

NOTE: Geoduck “pellets” were not very DNA pellet-like. Very loose, white, and sort of disintegrate (but not dissolve in solution) when attempted to resuspend.

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 yesterday was heat inactivated for 10mins @ 65C and 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 O/N in PTC-200 thermal cycler (MJ Research) – no heated lid.

Ligations will be stored @ -20C until I can continue working with them on Tuesday.

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

The previous attempt at making these RAD libraries failed during the prep-scale PCR, likely due to a discrepancy in the version of the Meyer Lab protocol I was following, so I have to start at the beginning to try to make these libraries again.

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 9.5μL:

Google Sheet: 20151028_RADseq_DNA_calcs

 

 

Prepared master mix for restriction enzyme reaction:

REAGENT SINGLE REACTION (μL) x11
DNA 9.5 NA
10x Buffer R 1.2μL 13.2μL
150μM SAM 0.8μL 8.8μL
AlfI 0.5μL 5.5μL

 

Combined 2.5μL of the master mix with 9.5μL of each DNA sample in 0.5mL snap cap tubes. Incubated @ 37C O/N in thermal cycler (PTC-200; no heated lid).

Troubleshooting – Oly RAD-seq

After the failure of the prep-scale PCR for the RAD library construction, Katherin Silliman pointed out a potential problem (too much dNTPs). This was odd because I was following the Meyer Protocol and I used what was indicated.

Oddly, it turns out that Katherine’s version of the Meyer Lab 2bRAD protocol differed from what I had download. To add to the confusion, both protocols have the same file name. Here’s what I’m talking about:

 

The file on the left is the one I was using and the one on the right is the file Katherine is using (NOTE: The file name’s aren’t exact because they were saved to the same directory and the numbers in the parentheses were appended to the file name automatically)

I’ve updated our copy of the protocol in our GitHub account. However, Katherine informed me that she’s just been pulling up the Meyer Lab page to reference the protocol. So, it’s possible they made a change to the file after I initially downloaded it, but the change wasn’t indicated in the file name.

http://people.oregonstate.edu/~meyere/docs/

However, when discussing with Katherine, she made a good point and said she just scaled up the test-scale PCR. Since the test-scale PCR was successful, she didn’t see a need to make any changes.

Will try this procedure again with the correct protocol; probably by scaling up the test-scale PCR.