Thank you for the wonderful visual+explanation! 15:53 May I know does the fragmentation occur before cDNA synthesis or after? I look through the protocol from Illumina they fragmented the RNA before cDNA synthesis
Thank you very much for that material! I had to perform RNA-seq but I didn't fully understand it and I was intimidating to ask my coworkers for explanation and you sir saved me! Your explanation is so clear and without rush. Thank you!
Hello there, We are really glad to hear that you found the video helpful! ;) Don't forget to check out other videos on our channel too! Thanks for watching :)
Thank you for this webinar. I have a question regarding the Quality Control before proceeding to the actual RNA seq. You have mentioned the Qubit, Bioanalyzer, and qPCR. Is it a step by step process or can use either one of this technology to assure the quality of the sample? Thank you in advance.
Each of these tools have different roles where Qubit is used to determine the RNA concentration in a sample. This is followed by using an Agilent Bioanalyzer to determine the RNA Integrity Number. The Bioanalyzer has a limited detection range and the Qubit value helps to determine if dilution is necessary prior to running on the Bioanalyzer. If your RNA concentration is usually within range, then the Qubit assay can be skipped. Once a library is prepped, library QC will also be performed using the Agilent Bioanalyzer to determine the library size and purity, and it also provides a Molarity value, though it is not always accurate. If no other downstream quantification method is available, this molarity value can be used for loading onto the sequencer. However, qPCR is highly recommended and is used to precisely quantify the library prior to loading the library onto the sequencer. Overloading or underloading can lead to failures in sequencing or poor quality data.
Very helpful and told the subtle differences. I have a question can we actually perform the differential expression fo the genes between the cancer samples (example- subtype or stage etc) without comparing them to control(non-cancer samples)? I appreciate your input
Yes, during the bioinformatics analysis stage, you can set up your comparisons such that you are comparing cancer to cancer samples or cancer to control samples. I.e. You can choose which set of samples to use for the comparison.
@@abmgood sorry I didn't clearly mention the question in my previous message. To rid off the somatic mutations we use the non cancerous sample control. But can we do it with out performing this as do the differential analysis of cancer to cancer?
Thanks for watching, Diego! To answer your question, if a transcript has no gene annotation, compared to the reference genome/transcriptome, this would likely represent a novel transcript. Keep in mind, though, that additional validation would be suggested for this.
@@abmgood Yes thank you very much. I am studying genomic medicine and a lot of the rocket scientist in my course are intimidating so this video is the most helpful video I have seen on rudimentary RNA-Seq and I will recommend it to co-students who are having trouble. My classes are RNA SARS Covid-19 I have a lot more confidence now.
I cannot stress ENOUGH how helpful this was. Thank you so much
Hello there,
We are really glad to hear that you found the video helpful! :) Thanks so much for watching!
Thank you for the wonderful visual+explanation!
15:53 May I know does the fragmentation occur before cDNA synthesis or after? I look through the protocol from Illumina they fragmented the RNA before cDNA synthesis
I struggled understanding RNA-seq but this presentation made it super helpful and easy to follow! Now I have a much better idea. Thank you!
Glad you found the video helpful! :) Thanks for watching!
Thank you very much for that material! I had to perform RNA-seq but I didn't fully understand it and I was intimidating to ask my coworkers for explanation and you sir saved me! Your explanation is so clear and without rush. Thank you!
Hello there,
We are really glad to hear that you found the video helpful! ;) Don't forget to check out other videos on our channel too! Thanks for watching :)
I've seen these topics being explained many times but never in such a clear and direct way. Thank you!
Thank you, Antonio! We are really glad that you found the video helpful ;)
I am more of a visual leaner and this greatly helped me. Massive thanks
Hello Bamu,
We are so glad to hear that you found the video helpful! :)
Thanks for watching!
Thank you so much for clarifying such a hard topic in a simple language. It was very helpful.
Glad it was helpful!
I want to know what (amplicon sequence variant) ASV data means. What do the headers ASV1, ASV2, ...etc. mean? Where can I find such explanation?
This is the best I have seen so far on RNA-seq... Thank you so much, sensei.
Glad it was helpful!
The best explanation of RNA seq! Much more clear than my lectures.
Thanks for watching! :) Glad you found the video helpful!
easily understandable and thank you for this this very informative video. is there any ways detect the circularRNA is QC step?
data analysis 22:00
Thank you for this webinar. I have a question regarding the Quality Control before proceeding to the actual RNA seq. You have mentioned the Qubit, Bioanalyzer, and qPCR. Is it a step by step process or can use either one of this technology to assure the quality of the sample? Thank you in advance.
Each of these tools have different roles where Qubit is used to determine the RNA concentration in a sample. This is followed by using an Agilent Bioanalyzer to determine the RNA Integrity Number. The Bioanalyzer has a limited detection range and the Qubit value helps to determine if dilution is necessary prior to running on the Bioanalyzer. If your RNA concentration is usually within range, then the Qubit assay can be skipped.
Once a library is prepped, library QC will also be performed using the Agilent Bioanalyzer to determine the library size and purity, and it also provides a Molarity value, though it is not always accurate. If no other downstream quantification method is available, this molarity value can be used for loading onto the sequencer. However, qPCR is highly recommended and is used to precisely quantify the library prior to loading the library onto the sequencer. Overloading or underloading can lead to failures in sequencing or poor quality data.
@@abmgood thank you very much. New subscriber here 🙂
oke bosss ku...
Hi this video was useful for me.thank you i just have a question what is the replicants mean in sequencing?
very nicely explained thanks a lot sir, but even after sign up i cant able to get ppt of this webinar please help me in this regard
thank you...
great
Thanks!
Is single cell sequencing the hottest spot in the current sequencing field？
Bergstrom Causeway
Happy learning. Thankyou 🙌
Happy to hear that you learn something from our videos! It's our pleasure!
This is the best introduction ever! thank you
Thanks so much for watching! :)
Wilford Forks
Andrew Shore
This is the best explanation on RNAseq. Subscribed and will watch more videos!
Thank you very much! This is so very helpful!
Hello Sage,
Glad you found the video helpful! ;) Thanks for watching and stay tuned for upcoming videos!
Excellent work. Thanks so much for sharing.
No problem! Thank you for watching:D
theng you.....boss
Thank you so much. My professor is a mess trying to explain this, and with this video I understood inmediatly.
Glad to hear that you found the video helpful! :)
A brilliant, clear and comprehensive video!
Thank you!
very nice information for bigginers.
Glad you found the video helpful! Thanks for watching!
Thank you !!!
Very helpful and told the subtle differences. I have a question can we actually perform the differential expression fo the genes between the cancer samples (example- subtype or stage etc) without comparing them to control(non-cancer samples)? I appreciate your input
Yes, during the bioinformatics analysis stage, you can set up your comparisons such that you are comparing cancer to cancer samples or cancer to control samples. I.e. You can choose which set of samples to use for the comparison.
@@abmgood sorry I didn't clearly mention the question in my previous message. To rid off the somatic mutations we use the non cancerous sample control. But can we do it with out performing this as do the differential analysis of cancer to cancer?
Great webinar, thanks a lot !!
How do I identify a novel transcript? For example, how would I know I discovered a new lncRNA??
Thanks for watching, Diego! To answer your question, if a transcript has no gene annotation, compared to the reference genome/transcriptome, this would likely represent a novel transcript. Keep in mind, though, that additional validation would be suggested for this.
prior knowledge of the gene (of course) that answers the question I had. tyvm
Hi there, thanks for watching and yes do let us know if you have any questions about the material presented!
@@abmgood Yes thank you very much. I am studying genomic medicine and a lot of the rocket scientist in my course are intimidating so this video is the most helpful video I have seen on rudimentary RNA-Seq and I will recommend it to co-students who are having trouble. My classes are RNA SARS Covid-19 I have a lot more confidence now.
Very good guide.
We're glad to hear that!
Very helpful, thanx!!
It was totally helpful to me, Thanx!
Thanks for watching!
THX
You're welcome! Thanks for watching.
Excellent explanation
Thanks so much! Glad you found the video helpful.
Great!
Thanks for watching! :)
Thanks alot
Thanks for watching!