Next Generation Sequencing 2: Illumina NGS Sample Preparation - Eric Chow (UCSF)

www.ibiology.org/techniques/n...
Next generation sequencing allows DNA samples to be sequenced quickly and affordably. Learn how next gen sequencing works and get tips on preparing and running your samples.
In the past decade there has been an amazing change in the efficiency of DNA sequencing. Using traditional Sanger sequencing, the human genome project took 20 years and cost $3 billion. Current next generation sequencing methods allow a human genome to be sequenced for $1000, in 48 hours! In this talk, Eric Chow explains the chemistry behind next generation sequencing, and describes how the next gen sequencers detect and display results. The most commonly used Illumina sequencers are image based and detect the addition of fluorescently labelled nucleotides. Chow also describes two different next generation sequencing technologies which provide benefits such as much longer reads but with downsides such as higher error rates. Chow finishes the talk with some insights into medical applications of next gen sequencing such as much less invasive prenatal testing or cancer detection.
In his second talk, Chow discusses Illumina NGS Sample Preparation. He goes over DNA and RNA preparation, bead-based (Ampure or SPRI) cleanups, and sample quantification and quality control.
And in two short how-to videos, Chow gives advice on purifying DNA samples using magnetic beads and on determining the quality of your nucleic acid sample using an Agilent Bioanalyzer.
00:00 - Start
00:36 - Review of next generation sequencing
02:13 - DNA library preparation
07:17 - RNA library preparation
14:26 - Bead-based cleanups
18:22 - Sample quantification and quality control
Speaker Biography:
Eric Chow is an assistant professor in the Department of Biochemistry and Biophysics and the Director of the Center for Advanced Technology (CAT) at the University of California, San Francisco. The CAT provides resources for UCSF labs wishing to use next generation sequencing techniques and Chow’s research program strives to develop new applications for NGS in pathogen diagnostics. Chow received his BA in molecular biology from the University of California, Berkeley and his PhD in biochemistry from UCSF.

[@Lenz979]

It is always a pleasure to listen to you Prof. Chow. Thank you so much for taking the time and doing these videos for us!

[@honeybunbadger]

You're the best, Prof. Chow! These videos are fantastic.

[@nicolaeionescu-kosa132]

It's a pleasure to listen to Prof. Chow

[@nehagoveas1]

Thank you! This was so wonderfully explained. Truly one of the best video out there!

[@DixOutForHarambe]

This is one of the few videos which accurately describes all needed details for NGS-beginners. Thank you so much dude! It is so much clearer for me now.

[@kmacdowe]

Complicated process explained simply. Thank you for the good work.

[@peterlauridsen8403]

This guy is so precise and thorough

[@angie800710]

Thank you for providing a well-explained tutorial video for the NGS beginner!

[@studybooks3395]

You are so smoking hooot!!!

[@sweeties6383]

it`s very informative . Nowadays I`m learning about sequencing . Thank you so much Professor.

[@NRoy877]

This is so simple and accurate, thank you for your video

[@mly2883]

Thank you Eric. Much needed video.

[@metipsm]

Excellent presentation. Thank you.

[@ccdj35]

You explained it perfectly. Thank you.

[@atrakchi1]

Well explained. Thanks!

[@joye.4281]

🤓Thank you soo much for this video! What I am doing now makes all sense. This is my first time working for a biotech company that also uses illumina sequencers. For someone who only worked in hospital core lab it was hard for me to understand biotech processes. This video helped me a lot and not just doing the steps in the lab without understanding. 💯 🙌

[@Tapj]

Thank you Eric!

[@cientistarj]

I've been learning so much with you, prof. Chow. Thank you so much!

[@zulucharlie5244]

Really great information - thank you.

[@mantidream8179]

Excellent explanation

[@llsa2009]

Very nicely explained video! Could you make video to explain different Lib Prep kits from various manufacturers with pros and cons?

[@chepai1827]

Nice talk!

[@shih-yihsiung239]

Thanks for the video.

[@dorisonuzulu9388]

Thank you SO MUCH!

[@isabelluo8752]

you are a true life saver!

[@rayzhang928]

these videos r so good

[@areejkhatib9095]

Amazing, thank you

[@desanonima]

Very nice, thanks!

[@bashiradilme]

your are awsome guys ...thank you

[@muhammadashrafhadirosman3845]

Good explanation :D

[@emavalenzuela8085]

Hi! Thank you for this video. I have a question, how meny samples can I sequencer in a single flow cell if I'm doing exome analysis. Thank you

[@DrApichatPhotiA]

It's very informative>>> You can simplify it. Thanks

[@kw-kt9xr]

If I had these videos 2 years ago I'd be getting a first for my first year instead of a low 2.1

[@devinyoung5735]

can you define what a "library" is in this context? Is it a term that comprises all of the fragmented genomic pieces?

[@1856chi]

Can you please do a video on Bionano?

[@Andrea-sh9sn]

Thanks a lot. What would happen if instead of putting 5% PhiX in the cartridge, I mistakenly put more amount (60%). And I put the correct amount from the library?

[@Philosophyof]

could someone tell me why are we using lower and upper marker?

[@merlindavid16]

I wish Prof. Chow taught me in college

[@user-wm8nn3gz7t]

As every target sequence has unique primers and PCR temperature.How to design PCR primers and protocol for amplification-based enrichment library preparation?

[@charlesw1973]

It's usually done by inorganic synthesis chemically and by other companies like IDT.

[@denizkarayagmurlu7162]

Can anyone please tell me, why 5' ends of fragments should be phosphorylated ?

[@sam2theammyk9]

13:48 I am so confused. How can you flip the orientation of DNA? I thought it was sequenced 5' to 3'? Are you sure the complement isn't being read? I don't see how the template can be read twice by flipping it because the orientation would then be 3' to 5'. That does not make sense to me. I'm so confused :(

[@charlesw1973]

It's actually still adding from 5' to 3'. It's just with the different indexes now The result strings looked flipped. Look up the illumina NGS video and you'll see the addition happens after the bridging step and is going from 5'3.

[@sarmadyousif2179]

Are all the DNA sequencing technologies need library preparation ? Yes or no ?

[@elleffeff]

so far, yes.

[@anhtruong2636]

the video image is too poor, you need to fix it more