Submitted 2bRAD Library 6 for Sequencing

I submitted another 2bRAD library today. This one has 25 samples of pooled Ostrea lurida larvae (and 3 samples from juvenile spat). Some of the samples correspond to the growth experiments done in Summer 2015, some of them correspond to the G2 South Sound oysters being used in Laura’s experiment, and some are just extras. The main goals of this library are to:

  1. See what pooled 2bRAD data “looks” like
  2. determine the amount of sequencing depth needed for confident genotyping (i.e. how many samples can go on a lane)
  3. determine if parentage can be assigned using 2bRAD sequencing from adults
    1. Bonus if successful- get an idea of how many parents contributed to my experiments

Details and notes from the library prep (including protocol) can be found at this Benchling note.

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Submitted 2bRAD library for sequencing!

Yesterday I submitted a 2bRAD library for sequencing at the UChicago Genomics Core, with an anticipated turn-around time of 1-2 weeks. The details of the library prep can be found at this Benchling note. At the top of the note there is also a tab for the protocol I followed, with the specific master mixes made for this library (one of my favorite features of Benchling).

FST and Admixture Ananlysis of 2bRAD Subset

I did a quick FST and Admixture analysis of the subset of 20 individuals from each population (19 from SS as one of them had inconsistent genotyping across replicates). You can view the results at this Jupyter Notebook on my github. These are very preliminary and will likely benefit from more individuals and by playing around more with the parameters in the mapping/genotyping. I plan to also look at nucleotide diversity.

 

 

Wednesday 11/25/15

Ran gels of samples B4-B10 from 2bRAD Library 1 of the PCR done on 11/24/15. Some of the PCRs either did not seem to work or might’ve washed out of the gel. I still cut out bands around 170bp for these, but will look at the quantification to see if anything is recovered. Gel slices were left in 4degC.

Samples that did not work (likely need redos): F5 8(HC1_4B), E5 (HC1_9), B5(HC2_13), B7 (HC3_9), B8(HC3_17, library 4).

 

Tuesday 11/24/15

Finished gel extraction of samples B3-C4 from 2bRAD Library 1 started on 11/23/15 following the protocol described in that post.

Did the PCR of the rest of Library 1 (B5-C12) with the new Taq. There is no H9, however (due to how I labelled on the spreadsheet).

First made more Lib 1 and Lib 2 primer stock:

  • 10 uL stock + 90 uL NFW = 10 uM

PCR master mix. Added 3.75 of each 1 uM barcode (HT and BC) to wells.

1x
65
10 mM (each) dNTPS
1.5
97.5
10 uM ILL-Lib1
1.5
97.5
10 uM ILL-Lib2
1.5
97.5
5X Q5 buffer
15
975
Q5 Taq polymerase
.75
48.75
20.25 + barcode = 27.75
648

 

Monday 11/23/15

Took the tubes with gel slices from Sunday 11/22/15 and centrifuged them at high speed for 1 minute to bring gel in contact with water. Put them in the -80degC freezer for 1.5 hours (Meyer protocol recommends at least 1 hour). Afterwards, centrifuged at maximum speed in the refrigerated centrifuge at 4degC for 15 minutes (Meyer protocol recommends 10-20 minutes). Then pressed gel slice against tube wall and took out between 30-50uL per sample and added then to a PCR plate corresponding to their well positions during the previous 2bRAD steps. I found that if the gel slices were left out of the cold for too long that they were more likely to break up during this step and less supernatant was recovered- in the future will work in batches of 16 and leave the rest in the fridge. Leftover gel slices were put back in the fridge in case they needed to be gel extracted using a kit due to low DNA recovery.

Ran out the gel of wells B3-C4 on a regular gel and cut out the band at around 170bp. Still need to do B4 from the PCR on 11/22/15. Left gel in 40 uL overnight at 4degC.

Gel_2bRAD_Lib1_B3_C4

Gel of samples B-C4 of 2bRAD Library 1

Did the Ligation 2bRAD step on libraries 2 and 3- decided to use 4 as a backup for failed individuals and put that plate in the freezer.

First made new adapters by adding the oligos into 2 separate tubes and leaving at room temp for 10 minutes:

Adaptor 1
150x 
5ILL-NR
7.5
Anti-ILL
7.5
NFW
735
Adaptor 2
150x
3ILL -NR
7.5 uL
Anti-ILL
7.5 uL
NFW
735 uL
Ligation master mix.
1x
149x
2 uM Adaptor 1
5 uL
745
2 uM Adaptor 2
5 uL
745
T4 ligase
1 uL
149
T4 ligase buffer with 10 mM ATP
4 uL
596
NFW
22
3278
10 mM ATP
1
149
Total
38
5662

Added 38 uL to each well.

11/21/15 and 11/22/15

Saturday 11/21/15

Added 8 uL of nuclease free water to Libraries 2, 3, and 4. Put in 4degC.

Sunday 11/22/15

Set up a PCR of wells H1-B4 of Library 1 (did not have enough Taq to do any more so ordered more Q5 High-Fidelity Taq). As the 100 uL PCR reactions recommended in the Meyer 2bRAD protocol always overfill my gel wells, I rescaled the recipe to be 77.75 uL reactions (50 uL ligation product + 3.75uL each barcode + 20.25 uL master mix).

First, made new working stocks of primers:

  • Made 10 uM stock of Lib 1 and Lib 2
    • 5 uL stock + 45 uL NFW
  • Made 1 uM stock of BC11, BC12, HT2, HT3, HT4, HT5, HT6, HT7, HT8
    • 1 sock + 99 NFW
1x
32
10 mM (each) dNTPS
2: 1.5
48
10 uM ILL-Lib1
2: 1.5
48
10 uM ILL-Lib2
2: 1.5
48
5X Q5 buffer
20: 15
480
Q5 Taq polymerase
1: .75
24
20.25
648

Added 3.75 uL of each barcode (this took a lot of time. I marked on the datasheet as I went to make sure I added barcodes to each well.)

Made a large gel with 24 wells and a regular gel with 8 wells. The top part of the large well broke so was only able to run 22 of the 31 PCRs (H1-C3). I put the gel slices in 1.5mL tubes with 40 uL of NFW and left overnight in the fridge.

Did the AlfI digestion of libraries 2, 3, and 4.

1x
230x
10x buffer R
1.2 uL
276
150 uM SAM
.8
184
AlfI (2 U/uL)
.5
115
water
1.5
345
4
920

Thermocycler at 37degC for 2 hours and 65degC for 15 minutes. Put in fridge afterwards.

11/19/15

DNA extraction of common garden broodstock:

  1. SS4_2b (redo of extraction attempted on 11/18/15)
  2. NF5_1
  3. NF5_2
  4. NF5_3
  5. NF5_4
  6. NF5_5
  7. NF5_6
  8. NF5_7
  9. NF5_8
  10. NF5_9
  11. NF5_10
  12. NF5_11
  13. NF5_12
  14. NF5_13
  15. NF5_14
  16. NF5_15
  17. NF5_16
  18. NF5_17
  19. NF5_18
  20. NF5_19

Afterwards, got the concentration of these extractions with Qubit. Plated of 1ug of DNA for Libraries 2, 3, and 4 (see “Libraries” sheet of the Common Garden Samples datasheet). Put Airpore tape over the plates and left in an incubator at 37degC.

11/18/15

DNA extraction of common garden broodstock. Eluted in 200 uL.

  1. NF3_17
  2. NF3_18
  3. NF3_19
  4. NF3_20
  5. NF4_1
  6. NF4_2
  7. NF4_3
  8. NF4_4
  9. NF4_5
  10. NF4_6
  11. NF4_7
  12. NF4_8
  13. NF4_9
  14. NF4_10
  15. NF4_11
  16. NF4_12
  17. NF4_13
  18. NF4_14
  19. NF4_15
  20. NF4_16
  21. NF4_17
  22. NF4_18
  23. NF4_19
  24. NF5_1
Did the ligation step for Plate 1 of the 2b-RAD broodstock libraries.
1st made fresh annealed adaptors.
Adaptor 1
96x 
5ILL-NR
5
Anti-ILL
5
NFW
490
Adaptor 2
11x
3ILL -NR
5 uL
Anti-ILL
5 uL
NFW
490 uL
Removed 5 uL of digestion product from each well to give 10 uL.
1x
96x
2 uM Adaptor 1
5 uL
480
1 uM Adaptor 2
5 uL
480
T4 ligase
1 uL
96
T4 ligase buffer with 10 mM ATP
4 uL
384
NFW
24
2304
10 mM ATP
1
96
Total
40
3840

Let to digest at 16degC for 2 hours followed by 10 minutes at 62degC and then put in freezer.

In the evening did another set of extractions. These are repeats of the ones done on 10/27/15 that turned out poorly.

  1. SS4_1
  2. SS4_2 (messed up)
  3. SS4_3
  4. SS4_4
  5. SS4_5
  6. SS4_6
  7. SS4_7
  8. SS4_8
  9. SS4_9
  10. SS4_10
  11. SS4_11
  12. SS4_12
  13. SS4_13
  14. SS4_14
  15. SS4_15
  16. SS4_16
  17. SS4_17
  18. SS4_18

When vortexing SS4_2 the cap wasn’t on all the way so most of it splashed out. Need to re-extract.

 

11/16/15 and 11/17/15

Monday 11/16/15

Took out the plate  from the incubator that was setup on Friday 11/13/15. The samples were completely dried down. I added 10 uL NFW to each well and left the plate in the fridge overnight. I also Qubit-ed more DNA extractions.

Tuesday 11/17/15

DNA extraction of broodstock from common garden experiment.

  1. NF1_9
  2. NF1_10
  3. NF1_11
  4. NF1_12
  5. NF1_13
  6. NF1_14
  7. NF1_15
  8. NF1_16
  9. NF1_17
  10. NF1_18
  11. NF1_19
  12. NF1_20
  13. NF1_21
  14. NF3_6
  15. NF3_7
  16. NF3_8
  17. NF3_9
  18. NF3_10
  19. NF3_11
  20. NF3_12
  21. NF3_13
  22. NF3_14
  23. NF3_15
  24. NF3_16

Eluted these in 200 uL to increase yield. Used Qubit to get concentration.

Set up a digestion of the 1st 2bRAD library plate. To save time, I did not take out 2 uL from each well to make 8uL as listed in the protocol. Instead I increased all of the master mix reagents so they would add up to 5uL, therefore maintaining a 2:1 sample:master mix ratio.

1x
96x
10x buffer R
1.2 uL -> 1.5
144
150 uM SAM
.8 -> 1
96
AlfI (2 U/uL)
.5 -> .625
60
water
1.5 -> 1.875
180
Total uL
5

Let digest for 2 hours at 37degC followed by 15 minutes at 65degC then put in the fridge.