Tag Archives: SNPs

Data Analysis – Subset Olympia Oyster GBS Data from BGI as Single Population Using PyRAD

Attempting to get some sort of analysis of the Ostrea lurida GBS data from BGI, particularly since the last run at it using Stacks crashed (literally) and burned (not literally).

Katherine Silliman at UIC recommended using PyRAD.

I’ve taken the example Jupyter notebook from the PyRAD website and passed a subset of the 96 individuals through it.

In this instance, the subset of individuals were all analyzed as a single population. I have another Jupyter notebook running on a different computer that will separate the three populations that are present in this subset.

Overall, I don’t fully understand the results. However, this seems to be the quickest assessment of the data (from the *.snps file generated):

28 individuals, 36424 loci, 72251 snps

Additionally, I did run into an issue when I tried to visualize the data (using the *.vcf file generated) in IGV (see screen cap below). I’ve posted the issue to the pyrad GitHub repo in hopes of getting it resolved.

 

One last thing. This might be obvious to most, but I discovered that trying to do all this computation over the network (via a mounted server share) is significantly slower than performing these operations on th efiles when they’re stored locally. Somewhere in the notebook you’ll notice that I copy all of the working directory from the server (Owl) to the local machine (Hummingbird). Things proceeded very quickly after doing that. Didn’t realize this would have so much impact on speed!!

Jupyter Notebook: 20160418_pyrad_oly_PE-GBS.ipynb

NBviewer: 20160418_pyrad_oly_PE-GBS

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HRMs – Lake Trout SNPs (HRM_white-05 & HRM_white_06)

HRM_white-05

The following primers from primer plate LTP01 will be used for analysis of Rick’s Lake Trout DNA1 plate (from 4/28/2009): G10, C11, H11, A12. HRM set up is here. It is the same as that used on 20090903. A 1:10 dilution plate (from 20090903) of Rick’s Lake Trout DNA1 plate (from 4/28/2009) was used. This means approximately 20ng of DNA used in each rxn. The robot was used to add 1uL of DNA from the 96-well plate to the appropriate wells of the 384-well HRM rxn plate. Plate was spun to collect the DNA at the bottom of the wells. The wells were visually inspected to ensure that each received the DNA. 1uL of DNA was manually added to those wells that did not receive sample.

The master mix for each primer set was then manually dispensed to the appropriate wells in the 384-well HRM rxn plate using a “matrix” auto-pipette. The real-time PCR was run in a Roche LightCycler480 machine with the following cycling parameters:

95C – 15mins

45 cycles of:

95C – 10s

60C – 15s

72C – 25s

After the completion of the real-time run, the plate was put through the High Melt Curve protocol.

 

 

HRM_white-06

The following primers from primer plate LTP01 will be used for analysis of Rick’s Lake Trout DNA1 plate (from 4/28/2009): E12. HRM set up is here. It is the same as that used on 20090903. A 1:10 dilution plate (from 20090903) of Rick’s Lake Trout DNA1 plate (from 4/28/2009) was used. This means approximately 20ng of DNA used in each rxn. The robot was used to add 1uL of DNA from the 96-well plate to the appropriate wells of the 384-well HRM rxn plate. Plate was spun to collect the DNA at the bottom of the wells. The wells were visually inspected to ensure that each received the DNA. 1uL of DNA was manually added to those wells that did not receive sample.

The master mix for each primer set was then manually dispensed to the appropriate wells in the 384-well HRM rxn plate using a “matrix” auto-pipette. The real-time PCR was run in a Roche LightCycler480 machine with the following cycling paramters:

95C – 15mins

45 cycles of:

95C – 10s

60C – 15s

72C – 25s

After the completion of the real-time run, the plate was put through the High Melt Curve protocol.

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HRMs – Lake Trout SNPs (HRM_white-03 & HRM_white-04)

HRM_white-03

The following primers from primer plate LTP01 will be used for analysis of Rick’s Lake Trout DNA1 plate (from 4/28/2009): E4, B5, A7, D7. HRM set up is here. It is the same as that used on 20090903. A 1:10 dilution plate (from 20090903) of Rick’s Lake Trout DNA1 plate (from 4/28/2009) was used. This means approximately 20ng of DNA used in each rxn. The robot was used to add 1uL of DNA from the 96-well plate to the appropriate wells of the 384-well HRM rxn plate. Plate was spun to collect the DNA at the bottom of the wells. The wells were visually inspected to ensure that each received the DNA. 1uL of DNA was manually added to those wells that did not receive sample.

The master mix for each primer set was then manually dispensed to the appropriate wells in the 384-well HRM rxn plate using a “matrix” auto-pipette. The real-time PCR was run in a Roche LightCycler480 machine with the following cycling paramters:

95C – 15mins

45 cycles of:

95C – 10s

60C – 15s

72C – 25s

After the completion of the real-time run, the plate was put through the High Melt Curve protocol.

 

HRM_white-04

The following primers from primer plate LTP01 will be used for analysis of Rick’s Lake Trout DNA1 plate (from 4/28/2009): H7, B8, C8, F10. HRM set up is here. It is the same as that used on 20090903. A 1:10 dilution plate (from 20090903) of Rick’s Lake Trout DNA1 plate (from 4/28/2009) was used. This means approximately 20ng of DNA used in each rxn. The robot was used to add 1uL of DNA from the 96-well plate to the appropriate wells of the 384-well HRM rxn plate. Plate was spun to collect the DNA at the bottom of the wells. The wells were visually inspected to ensure that each received the DNA. 1uL of DNA was manually added to those wells that did not receive sample.

The master mix for each primer set was then manually dispensed to the appropriate wells in the 384-well HRM rxn plate using a “matrix” auto-pipette. The real-time PCR was run in a Roche LightCycler480 machine with the following cycling paramters:

95C – 15mins

45 cycles of:

95C – 10s

60C – 15s

72C – 25s

After the completion of the real-time run, the plate was put through the High Melt Curve protocol.

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HRM – Lake Trout SNPs (HRM_white-02)

HRM_white-02

The following primers from primer plate LTP01 will be used for analysis of Rick’s Lake Trout DNA plate (from 4/28/2009): A3, D3, G3, A4. HRM set up is here. It is the same as that used on 20090903. A 1:10 dilution plate (from 20090903) of Rick’s Lake Trout DNA1 plate (from 4/28/2009) was used. This means approximately 20ng of DNA used in each rxn. The robot was used to add 1uL of DNA from the 96-well plate to the appropriate wells of the 384-well HRM rxn plate. Plate was spun to collect the DNA at the bottom of the wells. The wells were visually inspected to ensure that each received the DNA. 1uL of DNA was manually added to those wells that did not receive sample.

The master mix for each primer set was then manually dispensed to the appropriate wells in the 384-well HRM rxn plate using a “matrix” auto-pipette. The real-time PCR was run in a Roche LightCycler480 machine with the following cycling paramters:

95C – 15mins

45 cycles of:

95C – 10s

60C – 15s

72C – 25s

After the completion of the real-time run, the plate was put through the High Melt Curve protocol.

Results:

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HRM – Lake Trout SNPs (HRM-white-01)

The following primers from primer plate LTP01 will be used for analysis of Rick’s Lake Trout DNA plate (from 4/28/2009): A1, C1, H1, B2. So, that’s 4 primer sets x 96 DNA samples = 384. HRM set up is here. A 1:10 dilution plate of Rick’s Lake Trout DNA1 plate (from 4/28/2009) was made for HRM. This means approximately 20ng of DNA used in each rxn. The robot was used to add 1uL of DNA from the 96-well plate to the appropriate wells of the 384-well HRM rxn plate. Plate was spun to collect the DNA at the bottom of the wells. The wells were visually inspected to ensure that each received the DNA. 1uL of DNA was manually added to those wells that did not receive sample (B23, C23).

The master mix for each primer set was then manually dispensed to the appropriate wells in the 384-well HRM rxn plate using a “matrix” auto-pipette. The real-time PCR was run in a Roche LightCycler480 machine with the following cycling paramters:

95C – 15mins

45 cycles of:

95C – 10s

60C – 15s

72C – 25s

After the completion of the real-time run, the plate was put through the High Melt Curve protocol.

Results:

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qPCR – HRM Lake Trout SNP primer test

Tested out the plate of Lake Trout primers (LTP01 – forward and reverse combined) for SNP detection. qPCR was performed using Roche 2x HRM M.M. qPCR set up is here. Cycling params are as follows:

95C – 10mins

40 cycles of:

95C – 10s

60C – 15s

72C – 25s

Plate layout matches the primer plate layout.

Results: The following wells have signals and good, clean melting curves: A1, C1, H1, B2, A3, D2, G3, A4, E4, B5, A7, D7, H7, B8, C8, F10, G10, C11, H11, A12, E12. These are potential candidates for SNP analysis. Will test HRM analysis using these primers, each on a subset of Lake Trout DNA samples to see whether or not they’ll be truly useful for analyzing the full plate of DNA.

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