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Data Summary 2019 #40

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gwaybio opened this issue Dec 19, 2019 · 5 comments
Open

Data Summary 2019 #40

gwaybio opened this issue Dec 19, 2019 · 5 comments
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@gwaybio
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gwaybio commented Dec 19, 2019 •
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We are nearing the end of 2019, and we are starting to increase data collection. This issue will document the data we have currently collected.

Overall Summary

In summary, we've made a lot of progress in optimizing data collection, and a lot of the profiling results look promising. For example, we've identified some morphology features that are consistently different between mutant and wild-type clones. We can use the current data to finalize optimal conditions and optimal plate layouts so that we can efficiently scale up in 2020.

We can decide to scale up to profile different bortezomib resistant clones in HCT116. This would answer how many resistance mechanisms does this system develop. We could also scale up to test different proteasome inhibitors. The hypothesis there is that HCT116 cells develop the same resistance mechanism against any proteasome inhibitor. We could also scale to different cell lines, although this may be difficult since we'll have to optimize conditions again. The overall goal is to be able to predict resistance in single cells based on cell morphology. More exciting progress and questions to attack in 2020! 👀

cc @bethac07 @mekelley @shntnu @AnneCarpenter

At a Glance

HCT116 Cell Line (colorectal cancer), 2 treatments (DMSO control and Bortezomib (proteasome inhibitor)), 7 batches of data, 633 total profiles

batch_count

We have noted a potential issue in the number of cells in each well. High confluence may lead to incorrect segmentation and inaccurate profiles. This is one issue that we are working towards solving. I note the size of the single cell profiles (.sqlite file) for each plate. The bigger the size, the higher the confluence.

Batch Plate sqlite file size
2019_02_15_Batch1_20X HCT116bortezomib 11G
2019_02_15_Batch1_40X HCT116bortezomib 4.8G
2019_03_20_Batch2 207106_exposure320 7.2G
2019_06_25_Batch3 WTClones 23G
2019_06_25_Batch3 MutClones 26G
2019_11_11_Batch4 WTmut04hWed 56G
2019_11_11_Batch4 WTmut04hTh 56G
2019_11_19_Batch5 217755 37G
2019_11_20_Batch6 217762 28G
2019_11_20_Batch6 217760 48G
2019_11_22_Batch7 217768 17G
2019_11_22_Batch7 217766 39G

Traditionally, these files are between 10 and 25 GB in 384 well plates. These are only 96 well plates and the files are generally much larger.

Current Summary

Initial Testing

  • Batch 1 - tested magnification
    • We decided on 20X
  • Batch 2 - Replicate of batch 1 using only 20x data

Measure different clones

  • Batch 3 - Tested a bunch of different wildtype and mutation clones

Measure different time points

  • Batch 4 - Tested one extra day of growth (the cells in this batch might be too confluent to use)

Measure with lower confluence

  • Batch 5 - We tried to measure the same layout as batch 4 but with lower cell density

Test different confluence levels

  • Batch 6 - Testing two different plating densities
  • Batch 7 - Testing two different plating densities

Batch Details

Batch 1 - Acquired on 15 February 2019

Batch 1 data was acquired using two different magnifications (20X and 40X) (TODO see #28). The two batches are: 2019_02_15_Batch1_20X and 2019_02_15_Batch1_40X.

Notes - Batch 1

  • There were three cell lines tested: CloneA, CloneE, and WT.
  • The treatment tested was: Bortezomib
  • There were four doses tested: 0.0, 0.7, 7.0, and 70.0
  • The 0.0 dose represents 0.1% DMSO (control vehicle only)
  • Each profile was acquired in triplicate

Batch 2 - Acquired on 20 March 2019 (2019_03_20_Batch2)

Batch 2 data had the same data acquired as Batch 1. Batch 2 tested the same cell lines, the same perturbations, the same doses, and the same number of replicates as Batch 1.

Batch 3 - Acquired on 25 June 2019 (2019_06_25_Batch3)

Batch 3 data saw a shift in data collection. These cells have not undergone any treatment (not even DMSO/control vehicle). There were two plates (MutClones and WTClones). Each plate had many different wildtype and mutant clones. There were three wildtype parental lines acquired on both plates.

Notes - Batch 3

  • There were 18 mutant clones profiled: BZ001, BZ002, BZ003, ...
  • There were 15 wildtype clones profiled: WT001, WT002, WT003, ...
  • Wildtype parental profiles were collected on both plates
  • Profiles were acquired in triplicate

Batch 4 - Acquired 11 November 2019 (2019_11_11_Batch4)

Batch 4 also saw a shift in data collection. Cells were grown for 48h before fixation on both plates, however different densities were plated (10.5x10^3 cells/well for WTmut04hWed [plate #217744] and 7x10^3 cells/well for WTmut04hTh [plate #217748]). We've also now started collecting wildtype parental clones on every plate with 9 replicates.

Notes - Batch 4

  • These files are HUGE (56G) - there are too many cells here for the profiles to be reliable.
  • The plates were identical
  • Each profile was treated with DMSO or Bortezomib (7 nM)
  • Four mutant clones were tested (BZ001, BZ008, BZ017, BZ018)
  • Four wildtype clones were tested (WT002, WT008, WT009, WT011)
  • Wildtype parental lines were acquired on both plates, with both DMSO and bortezomib treatments
  • All profiles were captured in triplicate, except for wildtype parental lines treated with DMSO. These were collected 9 times.

Batch 5 - Acquired 19 November 2019 (2019_11_19_Batch5)

Batch 5 was the same experimental design as Batch 4. Batch 5 is a bit smaller than batch 4, but still very large (37G). Batch 5 was plated at 5x10^3 cells/well.

Notes - Batch 5

  • There is only one plate measured (217755)
  • Bortezomib and DMSO treatments
  • The same clones and replicate numbers as Batch 4

Batch 6 - Acquired 20 November 2019 (2019_11_20_Batch6)

Batch 6 was the same experimental design as Batches 4 and 5. There were two plates acquired in Batch 6: 217760 and 217762. We acquired brightfield images of these plates as well.

Notes - Batch 6

  • Bortezomib and DMSO treatments
  • The same clones and replicate numbers as Batches 4 and 5
  • Two different cell counts initially plated (217760 was 48G and 217762 was 28G)
  • Plated 5x10^3 cells/well in plate 217760 (imaged 5 channels)
    • Plate 217761 is the brightfield of plate 217760
  • Plated 2.5x10^3 cells/well in plate 217762 (imaged 5 channels)
    • Plate 217763 is the brightfield of plate 217762

Batch 7 - Acquired 22 November 2019 (2019_11_22_Batch7)

Batch 7 was the same experimental design as Batches 4, 5, and 6. There were two plates acquired in Batch 6: 217766 and 217768. Brightfield was also captured for this batch.

Notes - Batch 7

  • Bortezomib and DMSO treatments
  • The same clones and replicate numbers as Batches 4, 5, and 6
  • Two different cell counts initially plated (217766 was 39G and 217768 was 17G)
  • Plated 5x10^3 cells/well in plate 217766 (imaged 5 channels)
    • Plate 217767 is the brightfield of plate 217766
  • Plated 2.5x10^3 cells/well in plate 217768 (imaged 5 channels)
    • Plate 217769 is the brightfield of plate 217768

edits to add important details @mekelley described in #40 (comment)
@gwaybio gwaybio added the Data Data additions or descriptions label Dec 19, 2019
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gwaybio commented Dec 19, 2019

also cc @DavidStirling

@gwaybio gwaybio mentioned this issue Dec 19, 2019
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@mekelley
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mekelley commented Dec 20, 2019 via email

@gwaybio
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gwaybio commented Dec 20, 2019

@mekelley - It is also important to note that I reprocessed all replicate correlation plots in #42 (this includes updated batch 5 data, and new batch 6/7 data. I also removed costes (and other extreme outlier) features from all profiles. This made the profiles look much cleaner 🎉 We will continue dropping these types of features in future projects.

I haven't yet made a formal presentation of the updates, but noting here in case you wanted to take a quick look.

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Here are some additional notes regarding the 2019 year-end summary (I'll share the master spreadsheet after the holiday break since I'm still adding to it).

Batch 1:

  • The 0.0 nM bortezomib dose = 0.1% DMSO (control vehicle only)

Batch 3:

  • These cells had not undergone any treatment (not even DMSO/control vehicle)

Batch 4:

  • The bortezomib dose was 7 nM
  • Cells were grown for 48h before fixation on both plates, however different densities were plated (10.5x10^3 cells/well for WTmut04hWed [plate #217744] and 7x10^3 cells/well for WTmut04hTh [plate #217748])

Batch 5:

  • Plated 5x10^3 cells/well

Batch 6:

  • Plated 5x10^3 cells/well in plate #217760 (imaged 5 channels)
  • Plate #217761 is the brightfield of plate #217760
  • Plated 2.5x10^3 cells/well in plate #217762 (imaged 5 channels)
  • Plate #217763 is the brightfield of plate #217762

Batch 7:

  • Plated 5x10^3 cells/well in plate #217766 (imaged 5 channels)
  • Plate #217767 is the brightfield of plate #217766
  • Plated 2.5x10^3 cells/well in plate #217768 (imaged 5 channels)
  • Plate #217769 is the brightfield of plate #217768

Thanks again for all of your hard work. It's been a great year, looking forward to 2020!

@gwaybio gwaybio mentioned this issue Mar 10, 2020
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gwaybio commented Mar 10, 2020

Batch Heatmaps

Profiles generated with pycytominer (see #50)

Batch 1 (20X)

heatmap_2019_02_15_Batch1_20X

Batch 1 (40X)

heatmap_2019_02_15_Batch1_40X

Batch 2

heatmap_2019_03_20_Batch2

Batch 3

heatmap_2019_06_25_Batch3

Batch 4

heatmap_2019_11_11_Batch4

Batch 5

heatmap_2019_11_19_Batch5

Batch 6

heatmap_2019_11_20_Batch6

Batch 7

heatmap_2019_11_22_Batch7

@gwaybio gwaybio mentioned this issue Aug 6, 2020
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@gwaybio gwaybio mentioned this issue Nov 6, 2020
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