Grace’s Notebook: Bairdi RNA – Bioanalyzer and Nanodrop, Begin analyses for GSS – Kallisto

Today I tried doing several things, none of them super successfully. First, I tried using Kallisto to get some quick differential expression results from the new bairdi RNA-seq data. It didn’t work, but I just need to look a little more into it. I didn’t spend too much time on it. Secondly, I tried running 11 of the extracted RNA samples on the Bioanalyzer – things do not look good. Thirdly, I ran those same samples on the NanoDop. I am not sure what to make of the Nanodop results yet.

Kallisto

I made this GitHub Issue after meeting with Steven yesterday: #790

I tried using Kallisto. I downloaded it from here: https://ift.tt/2CmxB0C
I simply clicked the Mac link next to the most resent release date (10/4/2019).

I then tried using it in a jupyter noteboook: 2019-11-06-bairdi-kallisto.ipynb

Not entirely sure what there error message means, but I haven’t actually spent much time on it yet. Will look into tonight/tomorrow.

Bioanalyzer

These are the RNA samples I’ve chosen to create the 6 pooled transcriptomes:

FRP trtmnt_tank sample_day infection_status maturity tube_number total-yield_ng
6157 NA 9 0 M 169 603.2
6143 NA 9 0 M 10 380.9
6228 NA 9 0 I 29 335.4
6150 NA 9 0 M 119 323.7
6277 NA 9 0 I 177 296.4
6156 NA 9 0 I 24 275.6
6272 NA 9 0 I 111 260
6178 NA 9 0 M 124 215.8
6161 NA 9 0 M 53 201.5
6136 NA 9 0 M 91 192.4
6137 NA 9 1 I 81 403
6174 NA 9 1 I 20 360.1
6177 NA 9 1 I 54 327.6
6122 NA 9 1 I 133 305.5
6196 NA 9 1 I 170 300.3
6233 NA 9 1 I 117 300.3
6187 NA 9 1 I 158 241.8
6199 NA 9 1 I 151 241.8
6164 NA 9 1 I 71 236.6
6237 NA 9 1 I 17 226.2
6104 cold 12 0 I 259 412.1
6106 cold 12 0 M 241 169
6153 cold 12 0 M 209 315.9
6157 cold 12 0 M 224 249.6
6160 cold 12 0 M 238 494
6172 cold 12 0 M 316 373.1
6175 cold 12 0 I 246 269.1
6178 cold 12 0 M 218 162.5
6189 cold 12 0 I 216 577.2
6191 cold 12 0 I 227 234
6196 cold 12 1 I 203 546
6128 cold 12 1 I 231 492.7
6174 cold 12 1 I 233 457.6
6148 cold 12 1 I 213 442
6173 cold 12 1 I 210 432.9
6177 cold 12 1 I 250 410.8
6188 cold 12 1 I 245 409.5
6199 cold 12 1 I 254 369.2
6163 cold 12 1 I 228 364
6164 cold 12 1 I 257 353.6
6232 warm 12 0 M 286 663
6235 warm 12 0 I 297 531.7
6253 warm 12 0 I 275 387.4
6223 warm 12 0 M 270 380.9
6228 warm 12 0 I 290 370.5
6234 warm 12 0 I 263 345.8
6265 warm 12 0 I 282 317.2
6238 warm 12 0 M 375 301.6
6259 warm 12 0 M 281 300.3
6242 warm 12 0 M 377 289.9
6255 warm 12 1 I 262 536.9
6266 warm 12 1 I 264 526.5
6245 warm 12 1 I 365 481
6261 warm 12 1 I 273 469.3
6249 warm 12 1 I 279 461.5
6233 warm 12 1 I 371 370.5
6244 warm 12 1 I 284 358.8
6262 warm 12 1 I 289 352.3
6257 warm 12 1 I 363 269.1
6256 warm 12 1 I 283 247

I started out today by just working with the first 11 samples listed.

FRP trtmnt_tank sample_day infection_status maturity tube_number total-yield_ng
6157 NA 9 0 M 169 603.2
6143 NA 9 0 M 10 380.9
6228 NA 9 0 I 29 335.4
6150 NA 9 0 M 119 323.7
6277 NA 9 0 I 177 296.4
6156 NA 9 0 I 24 275.6
6272 NA 9 0 I 111 260
6178 NA 9 0 M 124 215.8
6161 NA 9 0 M 53 201.5
6136 NA 9 0 M 91 192.4
6137 NA 9 1 I 81 403

I made a chip all ready to go, but the computer and machine set-up was not great, so Sam helped fix it (connected laptop to monitor becuase laptop screen is too dark since it’s low on battery power).

I ended up losing that chip because it took 10+ minutes to fix the computer-machine situation, and you have to run a chip within 5 minutes.

So, I tried again:

I ran 1ul of each sample on a RNA Pico Chip. Gel:
img

Electropherogram:
img

After talking with Sam, he said that I should try running a blank plate to check that all the reagents work, but include the ladder.

Results from that:
Gel:
img

Electropherogram:
img

The ladder is not showing up (like it didn’t in the previous images with the samples), but the reagents are working correctly.

Then, since I had some gel-dye mixes I made that only last through today, we decided I should give those samples another try, but without the ladder. I don’t actually need a ladder for what I’m trying to do.

Results:
Gel:
img
Electropherogram:
img

I made a GitHub Issue with these results to get Sam’s input: GitHub Issue #792

Nanodrop

Per Sam’s suggestion, I ran those same samples on the Nanodrop (1ul each).

Results:

Sample ID User ID Date Time ng/ul A260 A280 260/280 260/230 Constant Cursor Pos. Cursor abs. 340 raw
10 Default 11/6/2019 4:47 PM 40.03 1.001 0.486 2.06 0.32 40.00 230 3.117 0.038
169 Default 11/6/2019 4:48 PM 51.50 1.288 0.599 2.15 1.92 40.00 230 0.671 0.020
29 Default 11/6/2019 4:49 PM 49.92 1.248 0.630 1.98 1.33 40.00 230 0.937 0.128
119 Default 11/6/2019 4:50 PM 33.50 0.838 0.486 1.72 0.22 40.00 230 3.826 5.695
119 Default 11/6/2019 4:52 PM 7.22 0.180 0.076 2.38 0.06 40.00 230 3.049 -1.480
177 Default 11/6/2019 4:53 PM 24.73 0.618 0.293 2.11 1.08 40.00 230 0.575 0.196
24 Default 11/6/2019 4:54 PM 26.51 0.663 0.319 2.08 1.13 40.00 230 0.588 0.025
111 Default 11/6/2019 4:55 PM 23.54 0.589 0.296 1.99 0.22 40.00 230 2.637 0.138
124 Default 11/6/2019 4:57 PM 15.28 0.382 0.170 2.24 0.57 40.00 230 0.675 0.016
53 Default 11/6/2019 4:59 PM 20.58 0.514 0.272 1.89 0.10 40.00 230 5.414 0.015
91 Default 11/6/2019 5:00 PM 15.70 0.392 0.201 1.96 0.19 40.00 230 2.022 0.621
81 Default 11/6/2019 5:01 PM 24.68 0.617 0.305 2.02 0.22 40.00 230 2.814 0.021
Sample ID Curve Type Ref conc Ref Abs Std 1 conc Std 1 Abs. Std 2 conc Std 2 Abs Std 3 conc Std 3 Abs Std 4 conc Std 4 Abs Std 5 conc Std 5 Abs Std 6 conc Std 6 Abs Std 7 conc Std 7 Abs
10 Interpolation
169 Interpolation
29 Interpolation
119 Interpolation
119 Interpolation
177 Interpolation
24 Interpolation
111 Interpolation
124 Interpolation
53 Interpolation
91 Interpolation
81 Interpolation

I also took screenshots of all the sample graphs: 20191106-notebook-images/nanodrop

from Grace’s Lab Notebook https://ift.tt/32mrunF
via IFTTT