My lab told me that the sequences are demultiplexed but the manifest file I generated from illuminating basespace have only sample ID and direction as in picture. How to know if data need demultiplexing or not? thanks
Hi Heba Huessin ,
The files are demultiplexed.
In order to understand Demultiplexing, frst you have to understand multiplexing.
When you perform NGS on a bunch of samples, unlike conventional methods, you combine all your samples into one and load it into the sequencer. The mixing up of samples (libraries) is "Multiplexing".
Now you may wonder how you will differentiate the samples after sequencing.
I assume you carried your NGS on the Illumina platform, so I will explain the process based on Illumina chemistry.
When you prepare libraries for NGS, you add two oligonucleotides to your DNA fragments - Adapters and index.
Adapters are oligonucleotides that enables your DNA fragment to attach to the flowcell.
Indexes (sometimes refers as barcodes) helps to differentiate your samples after sequencing.
For eg: Imagine you have 10 different samples, during library preparation you will add 10 different indexes to your 10 samples. Before loading on to sequencer you will combine (Pool) all the libraries together.
During sequencing, the index sequences are also sequenced.
After sequencing, the fastq file will contain all the sequences from your 10 libraries.
Now you have to "De-multiplex" the single fastq file to obtain the sequences from 10 different samples.
With a computational tool, you can sort all the sequences with the same index to a group and according to the index used you can assign the sequences to your sample.
So if you have separated sequences for all your samples, your sequences are demultiplexed.
Multiplexing is a wet lab process while demultiplexing is a computational process
Illumina provides indexes along with library prep kits, and some kits enable you to combine up to 96 samples for a single sequencing run.
I hope you got a clear idea now.
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