Question: Relationship between sequencing lane and ngs dataset
0
gravatar for m93
17 months ago by
m93170
m93170 wrote:

I am fairly new to NGS data analysis and I am struggling to understand the exact relationship between a sequencing lane and an NGS dataset. I should add I don't work in the lab, I only do bioinformatics.

I understand the basics of how to make NGS libraries (very briefly: shearing, ligation of adapters, optional PCR, ligation to solid surface, following by sequencing-by-synthesis or other method). What I am struggling with is the visualisation of what happens once you have made your libraries. My questions are:

  • If you have used adapters containing barcode sequences (that identify each patient), you would presumably end up with one single tube with your libraries in it right?

  • If so, you then "put" the contents of that tube along with DNA polymerase and nucleotides into one single lane of a flow cell? Your provider would then give you raw FASTQ files that have been identified using this barcode? So you would effectively received one (or 2, depending if it's single/paired-end sequencing) FASTQ file per patient right?

  • If you do not use adapters with barcodes, does that mean you would have to use a sequencing lane per patient? If so, that would vastly increase the price you pay your provider, would it not?

    Apologies if these are basic questions.

flow-cell lane ngs • 426 views
ADD COMMENTlink modified 17 months ago by genomax75k • written 17 months ago by m93170
2
gravatar for genomax
17 months ago by
genomax75k
United States
genomax75k wrote:

If you have used adapters containing barcode sequences (that identify each patient), you would presumably end up with one single tube with your libraries in it right?

Only if you pooled the individual libraries into one pool/tube.

If so, you then "put" the contents of that tube along with DNA polymerase and nucleotides into one single lane of a flow cell? Your provider would then give you raw FASTQ files that have been identified using this barcode? So you would effectively received one (or 2, depending if it's single/paired-end sequencing) FASTQ file per patient right?

You should get one (or two for PE) files per sample (patient) after demultiplexing. If you receive the data non-demultiplexed then one (or two for PE) files (for entire data) will contain all samples. You will need to separate them yourself.

If you do not use adapters with barcodes, does that mean you would have to use a sequencing lane per patient? If so, that would vastly increase the price you pay your provider, would it not?

Correct.

ADD COMMENTlink modified 17 months ago • written 17 months ago by genomax75k
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