Library Prep for NGS
The basic steps of a NGS library are as follows" 1.Sample extraction 2. DNA Fragmentation 3. End Repair and Adapter Ligation 4. Size Selection (varies according to different platforms chosen) 5. Library Amplification (by PCR) 6. Library QC (by qPCR, fluorometry, spectrophotometry and gel electrophoresis) 7. Library Pooling/ Multiplexing 8. Sequencing
Of course there are various cleanups and QC s being performed at different steps as necessary. At what step do we decide for depth and coverage (depends on applications such as whole genome or exome etc.),? Is there any calculation for Library pooling? Does above cover general library steps or is something missing?
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Hello Sujata!
We believe that this post does not fit the main topic of this site.
I suggest you read some manuals from e.g. Illumina for various kits that deal with these applications. These cover the library prep steps. Yes, there is a pooling calculator, please google for it, the first hit should be the one from Illumina itself. Depth and coverage is application-dependent, again please read manuals and tutorials which cover this. I encourage you to ask specific questions rather than "how do I do NGS" because the idea of this community is to help with specific problems (bioinformatic ones), so this question here is off-topic. Other more suitable communities could be ResearchGate, SeqAnswers or a suitable SubReddit.
For this reason we have closed your question. This allows us to keep the site focused on the topics that the community can help with.
If you disagree please tell us why in a reply below, we'll be happy to talk about it.
Cheers!
I agree to some extent but what about my specific question as to :
Depth and coverage are decided at the start of the experimental design but exactly at which stage of library prep is it implemented?
Please get a background in Illumina sequencing and you will be able to answer that yourself. Depth and coverage is determined by the number of reads and the read length and that again depends on the type of sequencer, the flow cell and the number of samples you pool. The fewer samples per run, the more reads per sample.