Molecular Minutes

Q&A: The Beginner's Guide to RNA-Seq Webinar

Posted by Applied Biological Materials (abm) on August 7, 2019

Here is the full list of answers to questions we've received during The Beginner's Guide to RNA-Seq webinar:

    1. Can you explain in a bit more detail why sequencing adaptors are used?
    2. Once the sequencing is done, how do I trim the adaptor sequence from the sequencing result? That is, how do I make sure the sequence information I have is for my target sequence, but does not include extra information, such as from the adaptors?
    3. What is the difference between gene profiling and gene expression?
    4. If I’ve done my experiment and have the raw sequencing data, but I do not know how to perform the analyses that I would like, can I give you the data and have you do the analysis?
    5. Do you know where I can get RNA seq data for cancer genomics research?
    6. Is it possible to sequence both circRNA and mRNA in the same RNA-Seq project? Is there an enrichment step or is this done through bioinformatics?
    7. How long does it take to do library prep, quality control, and sequencing?
    8. Can I submit one sample and use it for both RNA-Seq and miRNA-Seq? Or do I need to submit separate samples for those?
    9. Can I get one-to-one expert help if I want to have a deeper knowledge in all steps that are involved in sequencing?
    10. What happens if my samples fail QC?
    11. Can you explain a bit why clustering is necessary in the sequencing process?
    12. Do you have any representatives or distributors in Brazil or in the Czech Republic?
    13. How do I go about generating a heat map and principal component plot for my data? Is this something that you can help me with?

1. Can you explain in a bit more detail why sequencing adaptors are used?

The adaptor is important for the DNA fragment to bind to the sequencer; without this step, the sample would be washed away before the sequencing reaction begins.

The adaptor also includes a sequence which is important for the sequencing primer to anneal. Without this annealing, the sequencing-by-synthesis reaction could not occur.

Finally, when you sequence more than one sample at a time (called multiplexing), you need unique sequences in each adaptor to be able to distinguish Sample 1 from Sample 2 from Sample 3, etc. The adaptor sequence also contains this unique, sample-specific barcode, that allows you to tell which sequencing reads belong to which sample.


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Topics: Next Generation Sequencing, NGS, RNA-Seq

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