Friday 11/13/15

Starting to setup the 2b-rad libraries of adult broodstock that will be sent off in a couple weeks for sequencing. I made a sheet labelled “Libraries” on the Common Garden DNA Samples datasheet outlining which sample will go in which library. With our barcodes we can sequence up to 96 samples per lane. I decided to do about 75 per lane across 4 lanes, mixing up the 3 populations across the lanes. I’ll also repeat 3 samples across all lanes and repeat 2 samples within each lane.

I got the concentration of the extractions for NF2 with Qubit and then pipetted out 1 ug of DNA for the samples in Library 1 in a 96 well plate. I put Airpore tape over the plate and left it in a 37degC incubator to dry out.

 

Monday 10/19/15

Set-up a MiSeq run with the 2b-rad library (concentration of .815 ng/uL) finished on Friday 10/16/15. I’m using the MiSeq in the Pritzker DNA Lab at the Field Museum and a v3 reagent kit. To prepare the library for sequencing, I followed Illumina’s instructions. These instructions 1st call for a 4nM library, which I prepared based off these instructions: CALCULATIONSANDCONVERSIONSINPREPARATIONFORIlluminaMiseqRUN. The fragments should be approximately 176bp, so I used the following formula to get the nM conversion factor: 10^6/660/N (where N is the size of the fragment in basepairs). Taken from this website. This formula gave 8.6 which I rounded to 8. Therefore, to dilute my library:

  • 8nM*.815ng/uL = 6.5nM
  • Dilution: 6.5nM/4nM = 1.625 -> 1 uL sample + .625 water
  • Dilution x 5: 5 uL sample + 3.125 water = 8.125 total volume.

While preparing the library, Illumina gives options for the PhiX spike-in and the concentration to run on the flow cell. I chose a 2% PhiX spike in and 10pM as previous users of this MiSeq have reported overclustering at 11pM.

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

Wednesday 10/14/15

DNA Extraction of Oly Broodstock

  • SS1_1
    SS1_2 (not tan)
    SS1_3
    SS1_4
    SS1_5
    SS1_6
    SS1_7
    SS1_8
    SS1_9
    SS1_10
    SS1_11
    SS1_12
    SS1_13
    SS1_14
    SS1_15
    SS1_16
    SS1_17
    SS1_18
    SS1_19
    SS1_20
    SS3_1
    SS3_2
    SS3_5
    SS3_6
  • Found 2 tubes labelled SS1_2. Marked the unextracted one with a tan sticker
  • Could not initially find SS3_3 or SS3_4 but found later and reorganized box
  • Left in 60degC water bath for 3 hours
  • Eluted 50 uL then 100 uL

Primers

  • Received the rest of the 2b-RAD primers and resuspended them to 100 uM.
    • BC11
    • BC12
    • HT3
    • HT4
    • HT5
    • HT6
    • HT7
    • HT8
  • Made new 1 uM aliquots of BC2-10 and HT1
    • 1 uL stock + 99 uL NFW
  • Made new 10 uM  aliquots of Lib 1 and Lib 2
    • 2.5 uL stock + 22.5 uL NFW

PCR for 2b-RAD MiSeq run

Used 10 samples from 9/28/15 digestion and ligation. Numbers in parentheses are so I know which tube from the ligation each sample is in.

  1. SS2_6 (5)
  2. SS2_7 (6)
  3. SS2_8 (7)
  4. HC1_5 (8)
  5. HC1_6 (9)
  6. HC1_7 (10)
  7. HC1_8 (11)
  8. NF1_4 (14)
  9. NF1_5 (15)
  10. NF1_6 (16)

Master Mix

1x
10.5x
NFW
23 uL
241.5
10 mM (each) dNTPS
2
21
10 uM ILL-Lib1
2
21
10 uM ILL-Lib2
2
21
5X Q5 buffer
20
210
Q5 Taq polymerase
1
10.5
Added 50 uL of MM to 40 uL of ligation product. Added 5 uL of 1uM BC and HT to each sample, using BC1-10 and HT1.
Run out gel of 2b-RAD PCR product
This time, set up a medium gel for 8 of the samples and a small gel for the other 2. Made a 2% TAE gel, and after running it at 110V for 1 hour let the gels soak in water with 6 uL of EtBr to increase staining. PCR used 19 cycles.

Gel of 2b-RAD PCR product

Gel of 2b-RAD PCR product

20151015_124156

I think I should make sure to mix in the EtBr when doing the post staining as that is likely why there are bright white sections on the gels. It was still much easier to see the bands though. Cut out the bands and left them in 1.5 mL tubes in 4degC for gel extraction on Friday.

Monday 9/28/15

Do AlfI digestion of ethanol precipations from last week.
  1. SS2_3
  2. HC1_1
  3. NF1_1
  4. NF1_2
  5. SS2_6
  6. SS2_7
  7. SS2_8
  8. HC1_5
  9. HC1_6
  10. HC1_7
  11. HC1_8
  12. HC1_9
  13. HC1_10
  14. NF1_4
  15. NF1_5
  16. NF1_6
  17. NF1_7
  18. NF1_8
1x
20x
10x buffer R
1.2 uL
24
150 uM SAM
.8
16
AlfI (2 U/uL)
.5
10
water
1.5
30

Did ligation of same samples. Left for 2.5 hours at 16degC and did 10 minutes at 65degC following Matz protocol.

  • 1st make new adaptors.
  • Adaptor 1
    11x
    20x
    5ILL-NR
    .6 uL
    1.1
    Anti-ILL
    .6 uL
    1.1
    NFW
    58.8 uL
    106.91
    Adaptor 2
    11x
    3ILL -NR
    .6 uL
    Anti-ILL
    .6 uL
    NFW
    58.8 uL
    Master mix
    1x
    20
    2 uM Adaptor 1
    5 uL
    100
    2 uM Adaptor 2
    5 uL
    100
    T4 ligase
    1 uL
    20
    T4 ligase buffer with 10 mM ATP
    4 uL
    80
    NFW
    25
    500

    40 uL master mix + 10 uL digestion product

Wednesday 9/23/15

  • Did more extractions of broodstock samples. I think once this test library is done the broodstock libraries should be 1st priority, with samples from the stressor experiment 2nd and other larvae samples 3rd.
    • Used the EZNA kit with a 4 hour digestion time. All the tissue for HC1_16 was used up.
  1. HC1_11
  2. HC1_12
  3. HC1_13
  4. HC1_14
  5. HC1_15
  6. HC1_16
  7. HC1_17
  8. HC1_18
  9. HC1_19
  10. HC2_1
  11. HC2_2
  12. HC2_3
  13. HC2_4
  14. HC2_5
  15. HC2_6
  16. HC2_7
  17. HC2_8
  18. HC2_9
  19. HC2_10
  20. HC2_11
  21. HC2_12
  22. HC2_13
  23. HC2_14
  24. HC2_15
  • Finished the ethanol precipitation started on Tuesday 9/22/15. Left samples in 10 uL of water in 4degC overnight.
  • Qubit of samples for phylogeographic study. Made list of samples for GBS library.
      1. BC1_12: 25.2
      2. BC1_8: 6.44 (8)
      3. BC2_7: 10.6
      4. BC2_13: 11.5
      5. BC2_9: 13 (8)
      6. BC3_13: 108
      7. BC3_12: 15 (8)
      8. BC4_2: 8.84
      9. BC4_17: 11.4 (8)
      10. CA1_18: 24.4
      11. CA1_1: 36
      12. CA1_2: 17.4 (8)
      13. CA2_9: 43.8
      14. CA2_12: 18.8 (5)
      15. CA3_6: 62.3 (5)
      16. CA4_1: 5.1
      17. CA4_7: 19.8 (8)
      18. CA5_10: 48.8 (8)
      19. CA6_8: 5.14
      20. CA6_15: 25.2 (7)
      21. CA7_11: 7.4
      22. CA7_16: 19.1
      23. CA7_15: 14.9 (8)
      24. OR1_ 1: 7.66
      25. OR1_5: 29.2 (7)
      26. OR2_6: 12.9
      27. OR2_12: 15.8
      28. OR2_15: 18.2 (7)
      29. OR3_15: 9.24
      30. OR3_20: 13.7 (7)
      31. WA10_16: 25.4
      32. WA10_13: 9.52 (8)
      33. WA11_10: 18.7
      34. WA11_22: 17.4
      35. WA11_4: 11.7
      36. WA11_17: 4.78 (8)
      37. WA12_15: 24.4
      38. WA12_11: 4.18  (8)
      39. WA13_12: 7.2
      40. WA13_5: 4.4
      41. WA13_15: 45.1 (9)
      42. WA9_2: 11.4 (7)
      43. WA1_16: 62 (8)
      44. Conch_1: 12.4
      45. conch_2: 12.3
      46. conch_4: 9.65
      47. CA5_12: 21.8 (repeat with 2)
      48. CA5_10 repeat within

Tuesday 9/22/15

Lab Work

Set up a test PCR to determine the optimum # of cycles. You want to identify the minimum number of cycles required for a visible product at 166 bp. I chose 4 of the samples and tested them at 12, 17, 22, & 27 cycles as recommend in the protocol.

  • Made 100 uM stock of new ILL-Lib2 adaptor
  • Made 10 uM stocks of ILL-Lib1 and ILL-Lib2 primers
    • 1.5 stock + 13.5 uL NFW
  • Made 1 uM stocks of HT1 and BC1
    • 1uL stock + 99 NFW
1x
17x
NFW
4.6 uL
78.2
10 mM (each) dNTPS
.4
6.8
10 uM ILL-Lib1
.4
6.8
10 uM ILL-Lib2
.4
6.8
1 uM ILL-HT1
1
17
1 uM ILL-BC1
1
17
5X Q5 buffer
4
68
Q5 Taq polymerase
.2
3.4
Added 12 uL master mix to 8 uL ligation.
  1. SS2_3_12x
  2. HC1_1_12x
  3. NF1_1_12x
  4. NF1_2_12x
  5. SS2_3_17x
  6. HC1_1_17x
  7. NF1_1_17x
  8. NF1_2_17x
  9. SS2_3_22x
  10. HC1_1_22x
  11. NF1_1_22x
  12. NF1_2_22x
  13. SS2_3_27x
  14. HC1_1_27x
  15. NF1_1_27x
  16. NF1_2_27x

17 and 18 are 2 of the ligation reactions for comparison.

  • 17: SS_2_3
  • 18: NF_1_1
Programed PCR in thermocyclers 4,5,6. Called 2b12, 2b17, 2b22, and 2b27.
  •  (98°C 5 sec, 60°C 20 sec, 72°C 10 sec) X N cycles

9_22_15

It looks like 22 cycles is best, and worked on all samples so will be using that in the preparatory scale.

Ethanol precipitation

  • Set up an ethanol precipitation of all the broodstock samples I’ve extracted so far, as well as repeats of of the 4 samples used in the test scale PCR:
    • Population Sample Date extracted ng/uL Volume for 1 ug Volume sodium acetate Vol ethanol
      Oyster Bay BS_2_3 19.8 50.50505051 5.05 113.64
      Hood Canal BS_1_1 12.3 81.30081301 8.13 182.93
      Fidalgo BS_1_1 9.13 109.5290252 10.95 246.44
      Fidalgo BS_1_2 17.4 57.47126437 5.75 129.31
      Oyster Bay BS_2_6 23.4 42.73504274 4.27 96.15
      Oyster Bay BS_2_7 16.4 60.97560976 6.10 137.20
      Oyster Bay BS_2_8 16.8 59.52380952 5.95 133.93
      Hood Canal BS_1_5 12.1 82.6446281 8.26 185.95
      Hood Canal BS_1_6 23.5 42.55319149 4.26 95.74
      Hood Canal BS_1_7 14 71.42857143 7.14 160.71
      Hood Canal BS_1_8 22.8 43.85964912 4.39 98.68
      Hood Canal BS_1_9 13.9 71.94244604 7.19 161.87
      Hood Canal BS_1_10 27.7 36.10108303 3.61 81.23
      Fidalgo BS_1_4 8.9 112.3595506 11.24 252.81
      Fidalgo BS_1_5 18.1 55.24861878 5.52 124.31
      Fidalgo BS_1_6 16.1 62.11180124 6.21 139.75
      Fidalgo BS_1_7 35.8 27.93296089 2.79 62.85
      Fidalgo BS_1_8 22.7 44.05286344 4.41 99.12

      Left in ethanol and sodium acetate in -20C overnight.

Friday 9/18/15

Ligation of adaptors to digested samples.

Need to make new adaptors each time. Recipe below is for 10 samples (+1 for pipet error) to make adaptors at 2 uM.  Combine and let sit for 10 minutes at room temp – I made sure stocks were completely melted.

Adaptor 1
11x
100 um 5ILL-NR
.6 uL
100 um Anti-ILL
.6 uL
NFW
58.8 uL
Adaptor 2
11x
100um 3ILL -NR
.6 uL
100um Anti-ILL
.6 uL
NFW
58.8 uL
Ligation reaction master mix
1x
11x
2 uM Adaptor 1
5 uL
55
1 uM Adaptor 2
5 uL
55
T4 ligase
1 uL
11
T4 ligase buffer with 10 mM ATP
4 uL
44
NFW
25
275

*Note: the Meyer protocol calls for 1.0 uL rATP  and 4.0 uL T4 ligase buffer. My buffer had 10 mM rATP included, so I just used 25 uL of NFW instead of 24uL and did not add more rATP.

40 uL master mix with 10 uL of digested DNA. Held at 16degC for 2 hours and 15 minutes. Put in -20C.

Wednesday 9/16/15

2-B RAD Library Prep -Common Garden

Setup AlfI digestion of the 10 samples that were dried down and reconstituted on Monday following the Meyer Lab’s protocol. The SAM that was ordered was at .5 mM, so I made a dilution to 150 uM by adding 30 uL of stock to 70 uL of water.

1x
11x
10x buffer R
1.2 uL
13.2
150 uM SAM
.8
8.8
AlfI (2 U/uL)
.5
5.5
nuclease-free water
1.5
16.5
 Protocol calls for at least 1 hour at 37degC and up to overnight, so I set it for 2 hours followed by 15 minutes at 65degC.
Ran out gel of 2 uL digest and 1 uL of concentrated DNA. Meant to run out the digest next to the DNA for easier view, but forgot. I think I overloaded the DNA, will do .5 uL next time. What to expect:

  • “The original gDNA should appear as a single high-molecular weight band (>10kb) while the digested samples should be visibly degraded. An effective AlfI digestion produces a slight downward shift in the original HMW band and a subtle smear trailing downward from that band. If initial gDNA is degraded, this test is not useful and may be skipped.” -Meyer protocol
  1. SS_2_3 digest
  2. SS_2_4 digest
  3. SS_2_5 digest
  4. HC_1_1 digest
  5. HC_1_2 digest
  6. HC_1_3 digest
  7. HC_1_4 digest
  8. NF_1_1 digest
  9. SS_2_3
  10. SS_2_4
  11. SS_2_5
  12. HC_1_1
  13. HC_1_2
  14. HC_1_3
  15. HC_1_4
  16. NF_1_1
  17. NF_1_2 digest
  18. NF_1_2
  19. NF_1_3 digest
  20. NF_1_3
 gel_9_6_15
Some digests look good (SS_2_3, SS_2_5,HC_1_2,HC_1_3,NF_1_2, HC_1_4), some don’t look very degraded (SS_2_4) and some the DNA is degraded making it hard to tell (HC_1_1,NF_1_1,NF_1_3). For some reason NF1_3 dna does not show up at all- must have pipeted incorrectly. NF_1_1 and NF_1_3 looked degraded on the initial gel run, but weirdly HC_1_1 looked fine. I had to keep drying some of the samples after 10 minutes in the centrivap, which may have impacted DNA quality. Will talk to lab tomorrow, but my gut is to go ahead with library prep and sequence these on the MiSeq. If the 3 degraded samples have low read coverage, we’ll know how DNA quality effects library quality.