2) Next Generation Sequencing (NGS) - Sample Preparation

[@eunseo9745]

1. Whole Genome Seq (WGS) 1:20 1) TruSeq PCR-free Library Prep Kit 1:33 2) TruSeq Nano DNA Library Prep Kit 3:07 3) Nextera DNA Library Prep Kit 3:55 4) Nextera DNA XT Library Prep Kit 5:15 2. Exome Seq 5:35 1) Nextera Rapid Capture Exome Kit 6:14 2) Nextera Rapid Capture Expanded Exome Kit 7:40 3. RNA Seq 8:05 1) TruSeq Stranded Total RNA Kit 8:24 2) TruSeq Stranded mRNA Kit 10:04 3) TruSeq Small RNA Kit 10:24 4. Methylation Seq 11:15 1) TruSeq DNA Methylation Kit

[@abmgood]

Thanks for the timestamps, Ray! :)

[@ahmedalsaady3475]

Thank you Ray

[@dufo4766]

tip: the critical 3' adenylation of the fragments before ligation (dA tailing) is mediated by special enzymes, like the Ecoli DNA pol1 klenow fragment!

[@jlowry10001]

The quality of this video is fantastic! Well done.

[@abmgood]

+Josh Lowry Thank you! We are making the quality better with each new video, so be sure to checkout the rest!

[@dufo4766]

That was an excellent and very illuminating video, thank you Although you have to watch it several times to fully understand it!

[@abmgood]

Hi there! Thanks for leaving a comment - glad you enjoyed our video. If you want to learn more, feel free to check out our knowledge base: goo.gl/Ce0M4O

[@Biomeducated]

@DUFO476, ILLUMINAting... I see what you did there :D

[@huynamnguyen7174]

Thank for your interesting videos! Hi, I have a few questions that need urgent answers: 1/ How flourescent label in the blocking group are removed from the nucleotide? 2/ How to wash away free nucleotide? 3/ Why do we have to sequece 2 strain of DNA? 4/ What is the function of index 1 and 2 in adapter attached to DNA? Thanks you once again.

[@abmgood]

You’re welcome! 1) This paper (www.ncbi.nlm.nih.gov/pmc/articles/PMC4426941/) has more details on how the process works, but essentially it says that “A chemical deblocking step ensures the removal of the 3’terminal blocking group and the dye in a single step. The process is repeated until the full DNA molecule is sequenced.” 2) The sequencer washes away unbound nucleotides as part of the automated workflow. 3) Can you clarify what you mean by strain? 4) The indexes allow us to run multiple samples together and to identify samples during the DNA sequence analysis.

[@mimiwill5618]

Reply from an ordinary lay person: WAIT? WHAT?! 😂😂

[@TheCarbon14]

Great concise video, great information and way of presentation

[@abmgood]

Thank you for the nice comment and for checking out our video! :)

[@sharduldhole5701]

Very informative video! I have a few questions, 1. In Nextera Rapid Capture Exome Kit, How can we design oligos that are complementary to the exome without knowing the sequence of the exomes? 2. How the methylated and unmethylated cytosines that are converted to cytosine and uracil respectively are distinguished further during the sequencing step?

[@abmgood]

Thanks for watching! To answer your 2 questions: 1. The Illumina Nextera Rapid Capture Exome Kit is specific for the human exome. If you are working with a non-human species, this kit would not be suitable. In order to perform exome capture, you need to know the sequence of the exomes, so you can design appropriate oligos; if this information isn't available, you would likely not be able to design probes. 2. For Bisulfite/Methlyome-Seq, each treated sample is sequenced alongside an untreated sample, so that one can compare the two after to distinguish between converted vs. nonconverted cytosines. Illumna's website and Wikipedia have more detailed information regarding this if you're interested in further reading! www.illumina.com/techniques/sequencing/methylation-sequencing.html en.wikipedia.org/wiki/Bisulfite_sequencing

[@thanhtranhuynh2426]

Thank for your interesting videos! I have questions in RNA sequencing. 1/Why do we need to use UTP nucleotide instead of TTP nucleotide? 2/ After adapter ligation, we use enzyme to remove U nucleotide and my question is that Can we use DNA fragments which possess U nucleotide after deleting U nucleotide? Thanks you once again.

[@abmgood]

Hi Thành, thanks for your comment! The use of UTP is required for obtaining stranded sequencing information (i.e. ensuring that only the strand that is synthesized from the RNA is sequenced). UTP is only incorporated in the second strand synthesis step so that adapters can be ligated to the ends, and the UTP strand is removed and cannot be used afterwards.

[@sultanmia3895]

Nice ☺️

[@abmgood]

Thanks for liking

[@alicepat2750]

I really like your videos, just a note you spelled bioanalyzer wrong in a couple places(3:02, 10:04, and 11:10)

[@abmgood]

Hi Alice, thanks for watching and leaving a comment! Oops! Good catch!

[@biotechingenio]

Excellent video.I have a question about RNA-sequencing...at the point that you dismiss rRNA from your total RNA with magnetic beads (Ampure XP beads,maybe), how does this magnetic beads "recognize" the rRNA instead the other fragments of total RNA? I'm asking this because in my case I need that rRNA fragments to identify bacteria by 16S rRNA and indeed...I used Ampure XP beads in order to wash away that useless material (total RNA) and I don't understand completely how they work.Thanks for your attention.

[@abmgood]

Hi Jose, thanks for watching the video! The rRNA sequences are recognized by rRNA specific oligo probes. Once the oligo probes hybridize with rRNA sequences, they can be pulled down using magnetic beads. ​

[@believeinyourself7947]

Kindly suggest some good book name to study ngs in detail

[@yinjudy4489]

Great video! One simple question about the adaptor ligation (after adding A on 3’ of the blunt fragments), if I understand it correctly, the two ends of DNA fragments are supposed to link with different adapters right (the orange and green colour)? As both of them have the same 3'A, how to ensure that? I think fragments linked with the same adaptors will be produced at the same time right (orange and orange, green and green)? Do we do something specific to prevent this? Or we filter them out afterwards using the Bioanalyzer.

[@abmgood]

Thanks for your comment! It's easier to answer your question through a diagram: info.abmgood.com/hubfs/NGS-Adapters.png. Does this help?

[@yinjudy4489]

@@abmgood Yes that's very clear, thank you so much!

[@abmgood]

@@yinjudy4489 Glad to hear, and no problem! :)

[@dariocosemans8326]

What about fragments where the same adapter binds at each side? How do you make sure the fragments have two different adapters on each side?

[@martinmazukiewicz]

Important question for me. It's about the first Sars-Cov-2 ("WH Human 1", as the Chineses called it at that time. From the BALF taken from a patient, how could they create adaptors to catch the virus fragments as they didn't know its sequence yet ? They could't have primers. How the adaptors to catch an unknown RNA fragment can be built ? The only solution i can imagine : create four different primers with on each one one of the four single nucleotide (A,T,C,G). But is one nucléotique enough to fix the fragment ? Help !!! Thanks

[@abmgood]

Hello Martin, we will get back to you as soon as possible! :)

[@abmgood]

The first step involves RNA isolation from a human sample, followed by using a probe-capture technique to remove sequences from human nucleic acid. Many RNA viruses have poly(A) tails, including SARS-CoV-2, so the first step in library preparation takes advantage of this feature by using an oligo d(T) primer for the reverse transcription step to synthesize cDNA. Second-strand synthesis follows to generate double stranded DNA to use as template for NGS library prep. Library prep generally entails DNA fragmentation, end-repair, phosphorylation of the 5′ ends, adapter ligation, and PCR amplification for product enrichment. Following fragmentation, the DNA fragments are repaired to produce blunt-ends and 5'-phosphorylated ends compatible with the sequencing platform-specific adapter ligation strategy. This is done using a mixture of enzymes such as T4 DNA polymerase (for creating blunt ends) and T4 Polynucleotide Kinase (for 5' phosphorylation). The T4 DNA polymerase can blunt an end by either using polymerase activity to fill in the a 5' overhang or they can blunt a 3' overhang by degrading the overhang. This creates blunt ends that can be used for downstream adapter ligation (note: some sequencing platforms may also require polyA-tailing of the 3′ ends to facilitate ligation to the sequencing adapters). T4 DNA ligase can then ligate the adapters to the end-repaired library fragments, and the adapter sequences are used for PCR amplification prior to sequencing.

[@martinmazukiewicz]

@@abmgood Thank you so much for this detailed answer. I could never find such clear explanations on internet. You brought me all the information i needed. NGS is really, really fascinating. Thank you again. End of this problem. TTAGGGTTAGGGTTAGGG (DNA code for Telomere) ;-) Next one !

[@abmgood]

@@martinmazukiewicz No problem, Marin! :) Glad we could be of help!

[@KiranKampati]

good info video

[@mysticalgirl19]

very informative video, thank you :) How would you use NGS to identify Amyotrophic lateral sclerosis (ALS) with focus on RNA binding proteins TDP43, FUS, TAF15? What would be the approach?

[@abmgood]

Hi Julie! Thanks for your question and for your kind comments - we are glad you found the video helpful for you. Did you want to look for mutations in these selected genes? This sounds like a question our NGS helpdesk will be more equipped to answer, please feel free to email your questions to [e-mail].

[@raedahassan9166]

Background music is really noisy despite the video very useful

[@abmgood]

Hi Raeda, we've been working on fixing the loud background music in our newer videos. If it is very distracting, you can try watching it with the subtitles turned on and the audio muted since our videos are fully subtitled.

[@barbarad.1070]

which adapter ligates to the bead? the barcode-adapter or the P1-Adapter?

[@abmgood]

Hi Barbara, thanks for your comment! In exome preps, the genomic DNA is first "tagmented" (fragmented and tagged) using the Nextera transposase. Sequencing adaptors and barcodes are then added in a subsequent PCR amplification step. Next, the biotinylated target capture probes (probes that are complementary to the coding exons) are hybridized to the fragments, and the fragments that are bound to biotinylated probes are magnetically pulled down using streptavidin beads. After several wash steps to remove the unbound DNA, the fragments are eluted off of the beads. The adaptors do not ligate to the beads--the streptavidin beads are only used for magnetic pull-down of captured targets! Hope this helps--if you want more information about these methods, feel free to visit our knowledge base: goo.gl/W3ADIL

[@necranthemo]

Nice video but please enunciate!!

[@abmgood]

Hi Nikoletta! Thanks for your reply! We will certainly take your comment into consideration for our next videos!