Here is the full list of answers to questions we've received during The Beginner's Guide to RNA-Seq webinar:
- Can you explain in a bit more detail why sequencing adaptors are used?
- 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?
- What is the difference between gene profiling and gene expression?
- 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?
- Do you know where I can get RNA seq data for cancer genomics research?
- 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?
- How long does it take to do library prep, quality control, and sequencing?
- 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?
- Can I get one-to-one expert help if I want to have a deeper knowledge in all steps that are involved in sequencing?
- What happens if my samples fail QC?
- Can you explain a bit why clustering is necessary in the sequencing process?
- Do you have any representatives or distributors in Brazil or in the Czech Republic?
- 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.