Scope of Samples to Examine

[aspencoyle]

Alright, so I'm restarting much of my pipeline from the start, as I realized a flaw in my previous analysis (I was grouping Day 0 and Day 2 crabs at different temperature treatments, even though Day 0 samples were taken prior to exposure to different temperatures). The plan is as before: use kallisto to create a matrix of counts for each temperature group, then use DESeq2 to perform a differential expression analysis, then get the GO terms and use GO-MWU to perform a gene enrichment analysis.

The good news: This gives me a chance to recalibrate what libraries I want to include in my analysis of DEGs at different temperature treatments! I have 3 possibilities, as follows:

Option 1: Balanced Design

Temperature	Num of samples
Elevated	3
Ambient	3
Lowered	3

Option 2: Imbalanced Design
This option disregards a balanced design, and samples all possible infected crab from their respective treatment group

Temperature	Num of samples
Elevated	4
Ambient	10
Lowered	3

Option 3: Imbalanced Design - This time, it's imbalanced-er
This option adds some bonus ambient-temperature crab by including Day 0 crab that were part of the ambient and low-temperature treatment groups (and, since they were Day 0, had been held at ambient temperatures). This may be a plus or a minus, but this means that several individuals would be present in multiple temperature treatments (ex: Crab A is counted as an ambient library on Day 0, but a elevated-temperature library on Day 2)

Temperature	Num of samples
Elevated	4
Ambient	14
Lowered	3

Any recommendations on the best approach to take here?

[sr320]

These are individual crab libraries? And in some instances the same crab samples more than one? This would be a confounding factor. Can you provide a diagram of all libraries in play and how they relate?

On Tue, Feb 2, 2021 at 7:28 PM afcoyle ***@***.***> wrote: Alright, so I'm restarting much of my pipeline from the start, as I realized a flaw in my previous analysis (I was grouping Day 0 and Day 2 crabs at different temperature treatments, even though Day 0 samples were taken prior to exposure to different temperatures). The plan is as before: use kallisto to create a matrix of counts for each temperature group, then use DESeq2 to perform a differential expression analysis, then get the GO terms and use GO-MWU to perform a gene enrichment analysis. The good news: This gives me a chance to recalibrate what libraries I want to include in my analysis of DEGs at different temperature treatments! I have 3 possibilities, as follows: *Option 1: Balanced Design* Temperature Num of samples Elevated 3 Ambient 3 Lowered 3 *Option 2: Imbalanced Design* This option disregards a balanced design, and samples all possible infected crab from their respective treatment group Temperature Num of samples Elevated 4 Ambient 10 Lowered 3 *Option 3: Imbalanced Design - This time, it's imbalanced-er* This option adds some bonus ambient-temperature crab by including Day 0 crab that were part of the ambient and low-temperature treatment groups (and, since they were Day 0, had been held at ambient temperatures). This may be a plus or a minus, but this means that several individuals would be present in multiple temperature treatments (ex: Crab A is counted as an ambient library on Day 0, but a elevated-temperature library on Day 2) Temperature Num of samples Elevated 4 Ambient 14 Lowered 3 Any recommendations on the best approach to take here? — You are receiving this because you are subscribed to this thread. Reply to this email directly, view it on GitHub <#1093>, or unsubscribe <https://github.com/notifications/unsubscribe-auth/ABB4PN7FC6P6WBZLJI3Z4TDS5C7ERANCNFSM4XACMPXQ> .

-- Steven B. Roberts, Associate Professor Associate Director - Graduate Program Coordinator School of Aquatic and Fishery Sciences University of Washington Fisheries Teaching and Research (FTR) Building - Office 232 1140 NE Boat Street - Seattle, WA 98105 robertslab.info - sr320@uw.edu - @sr320 vm:206.866.5141 - cell:360.362.3626

[aspencoyle]

They're a mix of pooled and individual libraries (did my best to balance them out). And yep, absolutely - here's a table of the 3 possible sample situations. They are a bit large!

I can also post this as a separate question, but figured I'd ask in case it's a quick easy answer - looks like there are both raw .fastq files and fasta-trimmed .fastq files. Which should I be using as my initial data source?

Anyway, here are the tables!

Option 1: Balanced Design

Crab ID	Library ID	Day	Temperature at time
G	272	2	Elevated
H	294	2	Elevated
pooled	380825	2	Elevated
A	359	2	Ambient
B	349	2	Ambient
pooled	380820	0	Ambient
E	254	2	Lowered
E	445	17	Lowered
pooled	380823	2	Lowered

Option 2: Unbalanced Design

Crab ID	Library ID	Day	Temperature at time
G	272	2	Elevated
H	294	2	Elevated
I	280	2	Elevated
pooled	380825	2	Elevated
A	178	0	Ambient
A	359	2	Ambient
A	463	17	Ambient
B	118	0	Ambient
B	349	2	Ambient
B	481	17	Ambient
C	132	0	Ambient
C	334	2	Ambient
C	485	17	Ambient
pooled	380820	0	Ambient
E	254	2	Lowered
E	445	17	Lowered
pooled	380823	2	Lowered

Option 3: Mega-Unbalanced Design
Crabs appearing in multiple analysis groups are italicized

Crab ID	Library ID	Day	Temperature at time
G	272	2	Elevated
H	294	2	Elevated
I	280	2	Elevated
pooled	380825	2	Elevated
G	173	0	Ambient
H	72	0	Ambient
I	127	0	Ambient
A	178	0	Ambient
A	359	2	Ambient
A	463	17	Ambient
B	118	0	Ambient
B	349	2	Ambient
B	481	17	Ambient
C	132	0	Ambient
C	334	2	Ambient
C	485	17	Ambient
E	151	0	Ambient
pooled	380820	0	Ambient
E	254	2	Lowered
E	445	17	Lowered
pooled	380823	2	Lowered

In case you missed that, those were crabs G/H/I/E, sample numbers 173, 72, 127, 151

[sr320]

I would say go for all in terms of data exploration however none seem logical for an experimental design associated with a manuscript.

[aspencoyle]

Gotcha, I'll start on that. Can you elaborate a bit more on that - is there some configuration of the existing samples that would be manuscript-worthy, or would more sequencing be necessary?

Also for reference, the final table includes all existing libraries of infected crabs (both individual and pooled)

[sr320]

I see something worthy...

[aspencoyle]

I think I largely follow - comparison is between day and day 2 libraries for crabs G/H/I. Are the ambient-temperature crabs there to be used as a baseline for ambient-temperature expression - what's their purpose?

And that table does show all existing crab libraries! I can definitely work on prepping more for sequencing though, there are plenty of Day 2 elevated-temperature samples

[sr320]

I think I largely follow - comparison is between day and day 2 libraries for crabs G/H/I.

comparison is crabs ABC versus GHI, only focusing on two time points. To determine how temperature influences expression

And that table does show all existing crab libraries!

certainly have more pooled libraries? https://d.pr/i/MsOLkt

[aspencoyle]

Ahh okay, gotcha - I understand!

Although with the libraries: all libraries not included in that table are either uninfected libraries, which I assume are irrelevant for examining hematodinium response, or pooled by temperature (such as all those 329... samples sequenced by Sam), which don't seem useful for looking at temperature response.

Here's a photo from the spreadsheet that tracks the sample treatments specifically