What Is A "Lane" In Next Generation Sequencing Context?

Question

[Abdel]

What is exactly the definition of a lane in the context of sequence data? Many thanks!

Answer 1

[Damian Kao]

It refers to the physical lane on a flow cell that goes into the sequencing machine. Lanes might be used to separate different experiments or not.

[Madelaine Gogol]

A lane can also contain multiple samples if they are barcoded (with unique, short sequences for each sample).

[Abdel]

So how does that look as raw digital data? As a read? A sample?

[Leonor Palmeira]

You do not necessarily see a "lane" in your data. To put it simply, your sequencing provider should send you the data concerning your sample. You can sequence your sample on several lanes, for instance, but still get one file in the end. As well as sequence several samples on one lane as Madelaine explained and separate them after that according to their barcoding (small identical sequences attached to the read). Could you give more details on your question? Is it a general question, or concerning data you have received?

[Abdel]

Thank you, that was helpful. It was more of a general question. I kept running into the term in papers about processing sequence data, and did not understand what they meant.

[Abdel]

For example, Figure 1 in this paper: http://www.nature.com/ng/journal/v43/n5/full/ng.806.html

[Leonor Palmeira]

In that figure, they just analyse the reads by lane (probably each sample is on one lane only, which is the simplest experimental design).

[Rm]

http://www.cisd.ethz.ch/software/openBIS/Deep_Sequencing http://bioinformatics.oxfordjournals.org/content/25/17/2194/F1.large.jpg

[Madelaine Gogol]

I'm not sure if I understand.... Each read will have a barcode, different samples will have different barcodes.

Answer 2

[Sammy]

Hello. I'm here to read about lanes. I got my data from my sequencer provider with the file naming convention showing 4 lanes in total. Each experimental condition is found once in each lane. The way I'm reading this is that I have 4 replicates. Am I correct?

[lieven.sterck]

Hi Sammy ,

it is generally preferred to open a new question on the forum (and ideally link in this post/thread) than to add the question to an existing post. That way we can keep the forum better organized.

[GenoMax]

Each experimental condition is found once in each lane. The way I'm reading this is that I have 4 replicates

No you don't have 4 replicates. You just have data from 4 experimental conditions in each of 4 lanes.

[Sammy]

Thanks! So I have triplicates for four experimental conditions! That makes sense!!!

[Sammy]

I'd wish you were right but I've just been told that we have 4 replicates for each prep.

[GenoMax]

4 replicates for each prep.

What kind of prep? Do you have four biological samples that were made into independent libraries or do you have one sample that was prepped 4 times to make 4 libraries (technical library replicates)?

Consider following scenarios, if you do have 4 biological replicates:

• Those 4 replicates could have been pooled and them made into one single library that ran on one lane. In this case your may have started with 4 biological replicates but you can't tell them apart in data.
• It is certainly possible to create 4 libraries (with appropriate indexing scheme) and pool them into one pool that can run on one single lane. In this case you will demultiplex the data to recover 4 individual replicate data sets.

[Sammy]

Hey, GenoMax. I detailed the question here.