Molecular Minutes

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

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

RNA-Seq Webinar

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.


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?

For Illumina sequencing, the adaptor sequences are entered into the sequencer before sequencing; once sequencing is complete, the software can automatically trim the adaptors, filter this information out, and provide the FastQ files.


3. What is the difference between gene profiling and gene expression?

Gene expression can generally be thought of as pertaining to a single gene, whereas gene profiling refers to the expression level of many (or even thousands of) genes.

For all intents and purposes, though, gene profiling and gene expression can be considered to be the same thing.


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?

Yes, we have a dedicated in-house bioinformatics team that can assist with nearly any analysis you would need for your NGS project. Simply let us know the number of samples you have, the data format, and the type of analysis you are interested in, and one of our specialists can assist you further.


5. Do you know where I can get RNA seq data for cancer genomics research?

There are many publicly available data sets online where researchers have uploaded their RNA-Seq results.

For instance, NCBI has a Sequence Read Archive where researchers provide their data to the research community to ensure reproducibility and to allow other scientists to use their data set to make new discoveries or compare multiple data sets.

There are also other databases that exist, depending on the type of data set you are interested in, such as The Cancer Genome Atlas Program.


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?

For mRNA, a poly-A enrichment step is required. This step, though, will exclude transcripts that are not polyadenylated. To sequence circRNA, a special workflow is needed that selectively removes non-circular RNA prior to sequencing, as well as specialized bioinformatics. From standard RNA-Seq, circRNAs would generally not be detected.


7. How long does it take to do library prep, quality control, and sequencing?

For most RNA-Seq projects, our turnaround time is approximately 4-6 weeks, depending on the number of samples, amount of sequencing required, and sample/library QC. We also can offer expedited sequencing on a project-by-project basis.

The most lengthy part of the process is coordinating samples for a sequencing run, to ensure the samples will be efficiently sequenced, and provide a high-quality result.


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?

For RNA-Seq and miRNA-Seq different enrichment methods are needed to isolate mRNA or miRNA from Total RNA samples. Because of this, we would generally ask for two sets of samples to be submitted, one for each service.


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?

We’re very proud to offer quite a few resources for researchers that are interested in learning more. Our Knowledge Base and our YouTube channel both provide detailed information and step-by-step breakdowns of the sequencing workflows. For more detailed knowledge regarding library preparation and sample handling, Illumina has very detailed protocols that you may also find very helpful.

We also have additional webinars coming up that will provide more information regarding Whole Genome Sequencing and Amplicon Sequencing, although these webinars will be targeted towards beginners and people that are new to Next Generation Sequencing.


10. What happens if my samples fail QC?

Once we receive your samples, we will perform several rounds of QC, both before and after library prep. If at any point, your samples do not pass QC or we are able to identify any issues that may impact the ability to deliver a high-quality sequencing result, one of our specialists will reach out to you. They will then either ask if you can provide new samples or provide strategies for proceeding with your samples.


11. Can you explain a bit why clustering is necessary in the sequencing process?

With Illumina sequencing, the sequence is “read” by imaging the flow cell as the fluorescence signal from the newly-added nucleotide is emitted. A single fluorescent molecule is very difficult to reliably capture/image without super-high resolution imaging, and this is very difficult to do quickly and in real-time.

To address this, Illumina sequencing establishes a cluster of identical molecules that will then emit a small “cluster” of light. This is less challenging to reliably image/capture, and can allow for the rapid sequencing that is required for high-throughput next generation sequencing.


12. Do you have any representatives or distributors in Brazil or in the Czech Republic?

We currently have several distributors in both Brazil and in the Czech Republic. You can find their contact information on our Distributors page.


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?

The most important step for analyzing differences between samples is to normalize the data set so that samples can be compared to one another. For generating heat maps and principal component plots, for instance, the data must first be normalized using software, such as DESeq2. Once the data has been normalized, there are many different software packages that can be used to generate the figures, or you can even develop your own script to produce the visual data set.

Our in-house bioinformatics team can also help with this data processing and analysis, so that you have one less thing to worry about.


Got more questions? Feel free to contact our Technical Support Team at ngs@abmgood.com or leave your question in the section below. We'll be happy to help.

Topics: Next Generation Sequencing, NGS, RNA-Seq

Molecular Minutes

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For more in-depth articles, check out our knowledge base, which covers topics such as CRISPR, Next Generation Sequencing, PCR, Cell Culture, and more.

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