Question: A Simple Question On Rna-Seq Terminology
1
gravatar for alittleboy
6.0 years ago by
alittleboy210
USA
alittleboy210 wrote:

This question may be very simple and basic, but I just need to confirm that I understand the differences among those terminologies in the RNA-Seq context. Suppose I have a sample called SLR, and it is sequenced on 5 lanes, so I have (among other output files) BAM files like L1_SLR, L2_SLR, L3_SLR, L5_SLR and L7_SLR.bam. Here, the letter "L" denotes "lanes". In a typical RNA-Seq experiment, we should expect that reads from those 5 lanes have little variations, right? I am confused with the term "technical replicates" and the "lanes" here -- they're not the same, right? Also, I think these 5 lanes constitute a single "library", that is, one sample corresponds to one library. Am I right? That confused me because I once saw that each library corresponds to a technical replicate (e.g. mutant condition sample having 3 technical replicates, so it has 3 libraries; wild condition sample having 3 technical replicates, so a total of 6 libraries for the expriment). Thank you so much for your clarifications!

rna-seq library • 2.7k views
ADD COMMENTlink modified 6.0 years ago by JC8.0k • written 6.0 years ago by alittleboy210
5
gravatar for JC
6.0 years ago by
JC8.0k
Mexico
JC8.0k wrote:

A technical replicate is a single sample that is sequence again, Illumina's machines have 8 lanes to produce sequences, but each lane can hold more than one sample if required. Check this simple explanation here http://euler.bc.edu/marthlab/scotty/help.html

ADD COMMENTlink written 6.0 years ago by JC8.0k

thanks for the comments -- so in my situation, will the 5 lanes represent the 5 technical replicates? I am pretty sure that they come from the same sample, i.e. a sample that was sequenced on 5 different lanes. Also, how about the term "library"?

ADD REPLYlink modified 6.0 years ago • written 6.0 years ago by alittleboy210
3

They way you are describing your experiment, I do not think that the 5 lines are technical replicates. The page JC linked to is spot on. For the 5 lanes to be technical replicates, five separate aliquots "from the same solution" would have been extracted and prepared into a library independently of each other. If, however, a large aliquot was extracted and used to prepare a single library, which was then spread across five lanes, I would not call that a technical replicate.

I actually think your original interpretation of how you understand the terminology is correct, keeping in mind what JC pointed out that one lane can actually contain more than one sample/library through a technique called multiplexing so knowledge of the lane the data was captured from may not tell you much about the nature of the data itself.

ADD REPLYlink written 6.0 years ago by Steve Lianoglou5.0k
2

"will the 5 lanes represent the 5 technical replicates?" Possibly. "how about the term "library?" Library preparation is the process by which the RNA is fragmented and adapters are added so that it's ready for sequencing.

ADD REPLYlink written 6.0 years ago by Chris Miller21k
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