[BioC] Technical Replicates in EdgeR

[Neha Mehta]

Thank you for your answer! Moving forward I removed the lane that I
verified by plotMDS to be different from the other two. I have 2 further
questions.

1) I have a few highly expressed genes - the 2 most highly expressing genes
make up 23 and 10 percent of all mappable reads, respectively. Do I need to
do something to make sure that these genes will not have a negative effect
on my DE assessment? I plan to use edgeR for DE analysis, and I know I can
use TMM to normalize. Will this be enough?

2) When I ran a MAplot to compare my bio reps I saw that there are some
outliers, I have attached examples of 4 pairs of bioreps. Is this something
I should be concerned about?

Thank you again.

Neha Mehta


On Fri, Mar 21, 2014 at 5:03 PM, Ryan C. Thompson <rct at thompsonclan.org>
wrote:

> Hi there,
>
> No, there is absolutely no problem with dropping a lane of data if you
> believe that lane to have technical issues. I would recommend that you use
> the plotMDS function to verify that the counts in the bad lane are indeed
> different from the other two lanes.
>
> -Ryan
>
>
> On Fri 21 Mar 2014 01:45:51 PM PDT, Neha Mehta wrote:
>
>> Hello,
>>
>> I am a graduate student and fairly new to RNA-Seq. In my study I have a
>> 2x2
>> design with each group containing 4 bio reps and each bio rep has 3
>> technical replicates (same library prep, but each sample is processed in 3
>> different lanes). After studying my frequency count data I have found that
>> the count data in lane 1 is very different from lane 2 and 3 for most to
>> all bio samples. I plan to use EdgeR for DE analysis and I am wondering if
>> it is OK to sum count totals from just lanes 2 and 3. All documentation I
>> have read states that technical variation is small so you should sum all
>> tech reps, but in this case it seems I have noticed greater variation in
>> one lane and therefore it does not make sense to include it. I have not
>> found any reason why you can not just sum 2 out of 3 technical replicates
>> if you know that one technical replicate has much higher variation than
>> the
>> other two. Please let me know if you have any evidence for this.
>>
>> Thank you very much in advance.
>>
>>


-- 

Neha Mehta
---------------------------------------
PhD Candidate, Neuroscience
Northwestern University
ph. (412) 874-6342
e. nsmehta at u.northwestern.edu
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