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.

 

 

Size & 2bRAD Read #’s for MBD BSseq Samples

18 of the adult Olympia oyster broodstock samples that were 2bRAD sequenced were also sent off for MBD-enriched and bisulfite treatment sequencing to get epigenetic information (see project wiki). We wanted to associate size data with these samples and check if the 2brad sequencing worked.

First, some correction of sample names in the 2brad demultiplexing:

When I was on the last step of the 2brad protocol I accidentally deleted some of the sample names in Library 3 from the “Library” sheet in the master sample sheet. I didn’t notice until making the barcode sample sheet and so denoted those on the barcode sheet by putting “w” next to the name. I went back in the sheet revision history to 11/21/15 at 3:22pm and associated the correct name with the well and barcode for these and checked all other samples.

  • HC4_12w -> HC4_11
  • HC4_13w -> HC4_10
  • HC4_15w -> HC4_15
  • HC4_1w -> HC4_7
  • HC4_5w -> HC4_8
  • HC5_1w -> HC5_1
  • SS3_15w -> SS3_20
  • SS3_16w -> SS3_21
  • SS4_1Aw -> SS4_7
  • SS4_1Bw -> SS4_1
  • SS4_3w -> SS4_16
  • SS4_7w -> SS4_9
  • SS4_9 -> SS4_3

Size

When the broodstock oysters were dissected at Manchester last summer, I weighed them in the shell and took pictures of them first with a ruler so later they could be measured (dissections done on 8/13/15 and 8/17/15). I took the pictures for families HC1,  HC2, HC3, SS2, SS3, and SS5 and opened them in ImageJ. For each picture, I set the scale by drawing a line on 1 cm of the ruler to get the number of pixels per mm 3 times and getting the average pixels/mm. To get shell width, I drew a line between the two widest dorsal-ventral points of the shell (with the hinge always on bottom. I did this 2x for each oyster and took the average of the measurements.

Screenshot from 2016-02-12 16:28:19

I also measured shell area by drawing a line around the oyster shell 2x and taking the average. These obviously have more spread in the values.

Screenshot from 2016-02-12 16:33:26

Read Numbers

 

 

2bradbsanalysisb-0

2bradbsanalysisb-1

2bradbsanalysisb-2

Sample Weight (grams in shell) Length (mm) Raw 2bRAD reads
hc1_2B 2.2 17.41 1521
hc1_4B 1.9 20.43 2294290
hc2_15B 2.2 25.33 2498
hc2_17 1.1 19.38 (seq 4x)best = 2964986
hc3_1 2.2 26.79 1863108
hc3_5 1.9 19.5 1915
hc3_7 1.4 18.43 2438612
hc3_10 1.2 19.80 4091281
hc3_11 2.1 20.54 2901608
ss2_9B 1.5 17.2 749
ss2_14B 2.2 21.02 29745
ss2_18B 5.3 35.76 3016
ss3_3B 2.1 22.71 2076769
ss3_14B 2.4 24.71 2661152
ss3_15B 2.6 26.78 2816364
ss3_16B 4.2 39.57 2070520
ss3_20 2.3 26.96 1656456
ss5_18 5.4 27.34 2358172

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.