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Relationship between sequencing lane and ngs dataset
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Entering edit mode
23 months ago
m93 • 150

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.

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Entering edit mode
26 days ago
genomax 68k
United States

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.

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