Exploring GBS data and some file conversions

I’ve been working on organizing my scripts to analyze GBS data for my dissertation chapter on the phylogeography of the Olympia oyster. Still a lot of work to do, but here’s a couple of notebooks. All notebooks can be found at my Github under 2016_Notebooks and the scripts referenced in them can be found in my Github’s Scripts repository.

GBS_File_Conversions.ipynb: I’m planning on using this notebook to describe various file conversion scripts and code snippets I’ve written to work with. May end up splitting them into separate notebooks, but we’ll see. Currently, it describes how to add population information to the Structure (.str) output from ipyrad/pyrad, whether that be integers (required by the actual Structure program), or strings (useful for programs like Adegenet in R). It also describes how to take a .geno or .str file and split it up into many .geno files for looking at pairwise Fst and nucleotide diversity in R.

Exploring_GBS_Data_With_R.ipynb: This notebook has snippets of code used to explore pairwise Fst and nucleotide diversity (pi). Will eventually expand to include other R-based analysis methods.

 

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10/23/15

Common Garden Project

DNA extraction from Olympia oyster broodstock

  1. NF2_1
  2. NF2_2
  3. NF2_3
  4. NF2_4
  5. NF2_5
  6. NF2_6
  7. NF2_7
  8. NF2_9
  9. NF2_10
  10. NF2_11
  11. NF2_12
  12. NF2_13
  13. NF2_14
  14. NF2_15
  15. NF2_16
  16. NF2_17
  17. NF2_18
  18. NF2_19
  19. NF2_20
  20. NF3_1
  21. NF3_2
  22. NF3_3
  23. NF3_4
  24. NF3_5

Did not find NF2_8…

Olympia Oyster Population Structure Project

I started preparing genotype-by-sequencing libraries using the dried down samples from 10/21/15. Did an ApeKI digestion and ligation of adaptors.

Digestion:

1x 50x
NEB Buffer 3 2.μL 100
ApeKI 1 μL 50
H2O 17 μL 850

Added 20 uL of digestion master mix to each of the 48 wells. Incubated for 2 hours at 75degC then held at 4degC until ligation master mix was ready.

1x 51x
T4 Ligase Buffer 5 μL 255
T4 Ligase 1.6 μL 81.6 μL
H2O 23.4 μL

Added 30 uL of ligation mastermix to each well to give 50 uL total. Incubated at 19degC for 1 hour and 65degC for 20 minutes. Cooled to 0degC and put in freezer.

10/21/15

Common Garden Samples

  • DNA extraction of broodstock tissue. Let digest for 2.5 hours.
    1.  SS2_9
    2. SS2_10
    3. SS2_11
    4. SS2_12
    5. SS2_13
    6. SS2_14
    7. SS2_15
    8. SS2_16
    9. SS2_17
    10. S2_18
    11. S2_19
    12. S3_3
    13. S3_4
    14. S3_7
    15. S3_8
    16. S3_9
    17. S3_10
    18. S3_11
    19. S3_12
    20. S3_13
    21. S3_14
    22. S3_15
    23. S3_16
    24. S3_17

Running low on EZNA kit columns so ordered another kit.

Phylogeography Project

Setup dry down of 200 ng DNA from 48 samples.

BC1_12
BC1_8
BC2_7: 10.6
BC2_13: 11.5
BC2_9: 13 (8)
BC3_13: 108
BC3_12: 15 (8)
BC4_2: 8.84
BC4_17: 11.4 (8)
CA1_18: 24.4
CA1_1: 36
CA1_2: 17.4 (8)
CA2_9: 43.8
CA2_12: 18.8 (5)
CA3_6: 62.3 (5)
CA4_1: 5.1
CA4_7: 19.8 (8)
CA5_10: 48.8 (8)
CA6_8: 5.14
CA6_15: 25.2 (7)
CA7_11: 7.4
CA7_16: 19.1
CA7_15: 14.9 (8)
OR1_ 1: 7.66
OR1_5: 29.2 (7)
OR2_6: 12.9
OR2_12: 15.8
OR2_15: 18.2 (7)
OR3_15: 9.24
OR3_20: 13.7 (7)
WA10_16: 25.4
WA10_13: 9.52 (8)
WA11_10: 18.7
WA11_22: 17.4
WA11_4: 11.7
WA11_17: 4.78 (8)
WA12_15: 24.4
WA12_11: 4.18 (8)
WA13_12: 7.2
WA13_5: 4.4
WA13_15: 45.1 (9)
WA9_2: 11.4 (7)
WA1_16: 62 (8)
Conch_1: 12.4
conch_2: 12.3
conch_4: 9.65
CA5_13: 21.8 (repeat with 2)
CA5_10 (repeat within)

Keeping the calculations of uL to add on page 3 of this Google Sheet.

Friday 10/16/15

Common Garden 2b-RAD

Gel extracted 2b-rad PCR samples from Wednesday 10/14/15 using the Qiagen Gel Extraction Kit. Incubated 40 uL of elution buffer on the column for 5 minutes. Decided that going forward I would run PCR product out on a low melt gel and use gelase instead of the kit to save time/money.

Quantified gel extracted samples with High Sensitivity Qubit. Multiplied 37 uL by the lowest concentration to get ng per sample to add to pool, then calculated how many uL of each sample to add to pool. 37 * .373 = 13.8 ng.

Population Sample ng/uL after gel Vol to add to pool (uL)
Oyster Bay BS_2_6 0.907 15.21499449
Oyster Bay BS_2_7 0.388 35.56701031
Oyster Bay BS_2_8 0.661 20.8774584
Hood Canal BS_1_5 0.731 18.87824897
Hood Canal BS_1_6 1.45 9.517241379
Hood Canal BS_1_7 0.853 16.17819461
Hood Canal BS_1_8 0.96 14.375
Fidalgo BS_1_4 0.873 15.80756014
Fidalgo BS_1_5 0.373 36.99731903
Fidalgo BS_1_6 1.07 12.89719626

GBS Population Structure

Made new annealed adaptors for GBS as the current stock was over 2 years old. Followed Buckler lab protocol. buckler_lab_genotyping_by_sequencing_protocol_20110808