What is meant by the term "Library" in the context of wet lab & flow cell ?

[mallela]

Hi,

I was reading this paper and I came across the text,

"Because the most obvious source of variation between lanes is the differences in library size (i.e. sequencing depth), the simplest form of inter-sample normalization is achieved by scaling raw read counts in each lane by a single lane-specific factor reflecting its library size. "

Lets say, you have a plate of treated hela cells. Do you call this a Library ?

Now, you extract the mRNA and create cDNA from the above cells and distribute them on 6 lanes of flow cell (one chip) along with other bar coded samples. Do you call the reads in each lane as a different Library ? or do you call all the reads in one chip as one Library ?

Sorry for my naive question. I am just confused.

Thanks!

[IshSeq]

Hi,
A library is the fragments of nucleic acids that are that are tagged with indices (Also called barcodes).
Depending on the type of sequencing system you will be using and the sequencing depth required, you can add different number of libraries on the flow cell.

If you add only one library to the flow cell, then yes, all your reads are from one library. (Assuming your flow cell has only one lane)
If you add two libraries to the flow cell, then ideally you will be expecting the total reads from the flow cell to be split 50:50 i.e. half the reads from one and half from the other and so on.

As far as the Illumina systems are concerned, different machines have different flow cells. Also, the flow cells have varying number of lanes. You will have to look them up individually.

In this particular paper, I believe they have used a 8 lane flow cell and sequenced it on the HiSeq platform (A quick search found that they don't mention it anywhere, so your confusion is valid.) They have used one library/lane. Hence, they have chosen to call the number the reads obtained from one lane as the "library size" for that particular library

Hope that brings some clarity.

[mallela]

Thanks! That has cleared up some things for me.