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- เผยแพร่เมื่อ 21 พ.ค. 2024
- Next Generation Sequencing (NGS) is used to sequence both DNA and RNA. Billions of DNA strands get sequenced simultaneously using NGS. Whereas with Sanger Sequencing, only one strand is sequenced at a time.
While the Human Genome Project took over 30 years to sequence the human genome for the first time. Now with Next Generation Sequencing, a whole human genome can be sequenced in just one day.
This video describes the library preparation process, cluster generation, the sequencing reaction and filtering and alignment of the sequencing data. This video focuses on the primary sequencing method used by Illumina, Sequencing by Synthesis (SBS).
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CHAPTERS:
00:00 From the Human Genome Project to NGS
00:39 NGS vs Sanger Sequencing
01:01 The Basic Principle of NGS
01:23 DNA and RNA Purification and QC
01:38 Library Preparation - The First Step of NGS
02:32 Sequencing by Synthesis and The Sequencing Reaction
03:19 Cluster Generation From the Library Fragment
03:59 Sequencing of the Forward Strand
04:42 The First Index is Read
04:55 The Second Index is Read
05:15 Sequencing of the Reverse Strand
05:25 Filtering and Mapping of the Reads
05:47 Demultiplexing and Mapping to the Reference
06:19 What is Read Depth in NGS?
06:49 How is NGS being used?
07:01 What Types of NGS Applications Are There?
#ngs #sequencing #nextgenerationsequencing #ClevaLab
It doesn't matter how many times or how many videos I watch trying to understand how illumina works, I just can't wrap my head around that
Oh dear! Sorry to hear it.
@@ClevaLab 💀
I find it best to watch the videos on their website and TH-cam page. You could first tackle the tagmentation steps that show how the DNA fragments are fragmented and adapters ligated onto them. Then the clean up and amplification steps.
From there, there watch a video on sequencing by synthesis.
I don't think there's any one video or text that will fully explain the whole workflow. Best to break it up into small subtopics and fully exhaust each on it's own.
Hope I've helped😁
Same
you just have to watch a bunch of videos about it until you start piecing things together
4:25 FINALLY I GOT IT!! Everyone explains the detection like the fluorescence from the previous nucleotide just disappears. Thank youuuuuuuuuuuuuuuuuuuuuuu for eternityyyyyyy
So great! It makes a difference when you understand each step. 🙌 Thanks for your comment. 🤓
This video was an absolute gem!! Fantastic job, everything was well explained, illustrated and provided the base I needed.
Thanks for your comment. 🤓 So great! I'm trying to make my videos cover the basic principles of a topic, so I'm pleased to hear it gave you the base you need. 👍
The best NGS description video I've ever seen. It helps a lot! Thank you!
Thanks for your comment. I'm so glad it helped you. 🤓
Thank you for this video ClevaLab! I've been trying to understand Next Generation Sequencing for a project for a couple weeks now and this video helped all of it finally make sense.
I'm glad it was helpful. 🤓 Thanks for taking the time to comment. 👍
Best video on NGS out there. Beautifully done. Thanks
Thanks so much! That's great to hear. 🤓 I'm glad you liked it. Thanks for taking the time to comment. 🤗
Where was this video all my life!! Excellent explanation of the concept.
I'm so glad it was helpful. 🤓 Thanks for your comment.
Great job! Best explanation I found with all details needed. Thanks a lot!
Thanks for taking the time to comment. 🤓 I'm glad you're enjoying the videos.
This is freaking BRILLIANT, how have I not seen this channel before. I can't believe my luck in stumbling across it.
Subscribing RIGHT NOW.
Thank you, thank you, thank you.
You're so kind, thanks for watching. 🤓 You're lovely comment makes it all worthwhile! I'm so glad to have you as a subscriber. ❤
Can't wait for what you produce next. So greatful for your work, I understood this in less time than the 2 years I've been trying to understand this thing.
Just BRILLIANT 🥰
This is a great overview with really good visuals as well. Thanks so much. It’s very helpful for review as well.
Thanks for your comment. 🤓 I'm glad you found it helpful.
This ones amazinggggg! Best one on Illumina so far! Hope u would have elaborated more on how the terminator of dNTPs during sequencing works, and what exact enzymes (transposases) are used in DNA shearing. Thank you!
Everything is so cleanly done. This topic always confuses me but I feel much more confident thanks to you :)
Thanks for your comment. 🤓 Yay! I'm so glad it helped you.
Thank you for this one. It was what I have been looking for.
Thanks for your comment. 🤓 I'm so glad you liked it.
Your videos are amazing. They are clear, easy to understand, rich in contents, well illustrated and short. Perfect! Only a correction, the GHP complete 92% of the human genome instead of 85%.
I have done a lot of videos and this tutorial is the best ! Perfect for my internship in population genomics. Thanks a lot !
Thanks for your comment. 🤓 I'm so glad you liked it.
I have to say the video explains things better than my professor...😅
I'm glad it helped you. 🤓 Thanks for your comment. Maybe your professor should show the video in class!
Amazing illustration. Thank you.
Thanks for your comment. 🤓 I'm glad you like it.
Best illumina video ever!❤
Thanks for your comment. 🤓 I'm so glad you liked it. 🥰
💯
*Welcome to ClevaLab* - if you like the video, please give it a 👍 and subscribe for more videos. Also, if you have any questions, feel free to ask in the comments.
What an excellent explanation! Thank you!!!
Thanks for your comment. 🤓 You're very welcome, I'm glad you found it useful.
Absolutely amazing video!
Thanks for your comment. 🤓 I'm glad you liked it.
This was a really amazing explanation!! Instant Subscribe.... :)
Really loved it! Thank you so much for this gem!
Yay! Welcome to the channel. 🤓 I'm glad you enjoyed the video.
Beautiful animations, lovely video!
Thanks very much! 🤓 I'm glad you liked it.
Best video for NGS study so far
I'm glad you liked it. 🤓 Thanks for your comment.
THANK YOU! This is excellent!
Glad you liked it! 🤓 Thanks for your comment.
Fantastic tutorial!
Thanks for your comment. 🤓 I'm so glad you liked it.
Thanks ClevaLab!
I'm an high school student. And I'm supposed to write an essay about NGS for the school club. Thanks to these explanations and animations I could understand how it works. Cheers!
That's great to hear, I'm so glad. 🤓 Thanks for taking the time to comment.
Amazing. Thank you so much 💓
@sonalvishwakarma30 I'm glad it was helpful. 🤓 Thanks for your comment.
Great work!
Thanks for your comment. 🤓 I'm glad you liked it.
so helpful, thank you so much!
Thanks for your comment. 🤓 I'm so happy you liked it.
Excellent educational material
Thanks for your comment. 🤓👍
Thank you!❤
You're welcome! 🤓
Thank you!
I'm glad you liked it. Thanks for your comment. 🤓
This is perfect!
Thanks for your comment. 🤓 I'm glad you found it helpful.
This was an excellent presentation - I’ve been trying to find a succinct and great explanation of NGS in relation to the industry I work! Subscribed!
If you honestly made a video of every single individual function of how NGS is used (the last animation in the video), that would be so incredibly useful!
Thanks for your comment, Moses. 🤓 I'm so glad you found it so helpful. Great suggestion. I do plan to do more specific videos on NGS applications in the future. Stay tuned! Thanks for subscribing. ❤
great Video, i love it
Thanks for your comment. 🤓 I'm so glad you loved it.
Beautiful
Thank you. 🤓 I'm glad you liked it.
Entendí una gran parte, graciasss
Thanks for taking the time to comment. 🤓 I'm glad you're enjoying the videos.
you are a genius!!!!
Thanks for your comment. 🤓 I'm so glad you liked it. 👍
excellent
Thanks for your comment. 🤓 I'm so glad you liked it.
great video
Thanks for your comment. 🤓 I'm glad you liked it.
It's too Hard for me to understand ... I'm re-watching thousands of time .. but you explained so well .. thanks.
Thanks for your comment. 🤓 Yes, it's aimed at people with prior knowledge of some biochemistry. Do you have any questions that I can help clear up?
Awesome
Thanks for your comment. 🤓 I'm so glad you liked it.
Thank you🥹
You're welcome. Thanks for your comment. 🤓
Can I find the sources anywhere? Great video!
I loved this video, it was really helpful. I was once told how many wells were in the flow cell and how many clusters in a well. But my notes on my phone where I typed it in the moment (I was on a tour) didn't save. Does anyone know these answers?
Thanks for your comment and question. 🤓 The number of lanes per flow cell depends on the instrument. The iSeq 100, MiniSeq, MiSeq, NextSeq 550 and Next Seq 1000/2000 all have flow cells with a single lane. The NovaSeq 6000 has several flow cells - the SP, S1 and S2 all have 2 lanes, and the S4 has 4 lanes. The HiSeq 3000/4000 and NovaSeq X all have 8 lanes per flow cell.
The cluster density is a range for the non-patterned flow cells of the MiniSeq, NextSeq 550 are given in this link: knowledge.illumina.com/instrumentation/general/instrumentation-general-reference_material-list/000001511
The iSeq 100, NextSeq 1000/2000, NovaSeq 6000, HiSeq 3000/4000 and NovaSeq X all use patterned flow cells, so they have a fixed cluster density. For these, you look at the % of clusters passing the filter. They have millions to billions of clusters depending on the instrument.
The whole concept i get is , "then the nucleotides are washed away" 😂
Ma'am your explanation was too good n clear, could you pls tell how to do phd in abroad in biotechnology
Thanks for your comment. 🤓 I'm glad you liked it.
The animation is so good for learning this stuff. Thank you. However, you don't need reference genes or sequences to assemble libraries of reads; de novo assembly doesn't need them (e.g., de Bruijn graphs).
Thanks for your comment. 🤓 Sure, but this video is intended to give a basic intro to DNAseq and NGS.
Great video! I wonder who the HGPs DNA comes from. How do we know it is representative of all humans, now that this new technology depends on it.
Thanks for your comment. 🤓 That's a great question. The *DNA for the Human Genome Project came from 12 anonymous donors* . DNA from 9 of the donors were used in the final assembly. The samples were de-identified entirely from the name as well as the ethnicity of the donors. Not all samples were used in the final assembly, so not even the donors know if their DNA is in the sequence.
Interestingly, *74% of the original human reference genome is from one donor (RP11)* . It was later determined that this donor was likely African American and of mixed African and European ancestry. (journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1000360).
However, the Genome-Wide Association Studies (GWAS) that followed the Human Genome Project were largely of European ancestry (78% European, 10% Asian, 2% African, 1% Hispanic and 1% Other). This lack of diversity sparked an international project called the 1,000 Genomes Project. It sequenced samples from Europe, East Asia, sub-Saharan Africa and the Americas. This project sequenced over 1,000 people by 2012.
But, the human reference sequence is still 70% from one donor (RP11). To increase the diversity of the human reference sequence. The Human Pangenome Project was started. They aim to create 350 complete human reference genomes from diverse genetic backgrounds. As of 2022, a draft Human Pangenome Reference from 47 genomes is available (www.biorxiv.org/content/10.1101/2022.07.09.499321v1).
Quick question regarding Adaptor Dimers. Those can also compete with your library to bind to the flow cell and take up reads right?
Thanks for your question. 🤓 That's correct. 👍 If you don't remove the adapter dimers, they'll also bind to the flow cell. If you only have a small amount of adapter dimers, it shouldn't be too much of an issue. But you'll lose some of the sequencing capacity of the flow cell by sequencing the adapters.
Please let me know if you have further questions.
I really enjoy this video, but I have a question how the adapters bond to the different fragments of DNA?
Thanks for your comment. 🤓 That's a good question. The method for adding adapters to the DNA fragments will depend on the library prep kit. Either PCR, ligation or tagmentation is used to add the adapters. With PCR, the adapter sequences are included in the primers used to amplify the DNA. With ligation, DNA ligase sticks the adapters onto each end of the already fragmented DNA. And finally, with tagmentation, a transposome is used to fragment the DNA and add the adapters.
Lot of progress since I took biochemistry in 1970.
Yes, there has been! Thanks for your comment. 🤓
Do you also have videos about the bioinformatics part?
Thanks for your question. No, not yet, but it's definitely on the list!
No points for error in this approach at all :)
Thanks for your comment. 🤓 Do you mean errors in the sequencing or just generally?
Generally you covered a multi step process very well
Thank you so much for this knowledge full video,
Please answer a question.
If we have genome size of bacteria 2.3 MBp then what will be the library size for whole genome.
If some video available for whole process please do share. And please give answer of this question.
Thanks once again
Thanks for your question. 🤓 For bacterial DNA whole genome sequencing, the DNA is cut into pieces and adapters are attached to form a library, just as for human DNA. The library size will be around 500 bp for a 350 bp insert size. There are not many easy-to-understand videos or guides out there, which is why I started this channel! You can look at the Illumina website for more on microbial sequencing by NGS. www.illumina.com/areas-of-interest/microbiology/microbial-sequencing-methods.html
@@ClevaLab thank you 😊🤗, you are doing very great 😃.
Do the dna sequencers have all the reagents in the casette?
Thanks for your comment. 🤓 Yes, generally, the reagents come in pre-filled reagent cartridges that get loaded onto the instrument. The cartridge types and amounts vary depending on the sequencing instrument. There can be one reagent cartridge, several, or additional bottled reagents. You can see the different Illumina instrument sequencing reagents here: sapac.illumina.com/products/by-type/sequencing-kits/cluster-gen-sequencing-reagents.html
why reverse strand is washed off ??? @ClevaLab
Great video, but what if the dna sample is from an unidentified organism, or to put it simply what if we have no reference genome ?
Thanks for your comment. I'm so glad you liked it. 🤓
Sequencing a genome from a new organism is called _de novo_ sequencing. You can perform _de novo_ sequencing using NGS, Nanopore sequencing or single molecule real-time (SMRT) sequencing (from PacBio). Short or long reads can be used. The bioinformatics software uses overlapping segments of DNA reads to create the genome assembly.
In the past, after Sanger sequencing, the Human Genome Project used overlapping fragments to assemble the human genome. However, now sequencing instruments can sequence much more DNA per day. The bioinformatics utilised to perform the assemblies have also improved dramatically. I've just uploaded a new video on Sanger Sequencing, so feel free to check that one out as well. (🔗th-cam.com/video/X9566yI2cBo/w-d-xo.html).
Please let me know if you have further questions.
Can you explain more about adapters please?
Thanks for your question. 🤓The adapters contain a region that can bind to the oligo on the surface of the flow cell, an index, and a region to bind the sequencing primer. The index identifies the sample.
You can see an image and read more about them here: knowledge.illumina.com/library-preparation/general/library-preparation-general-reference_material-list/000003275
@@ClevaLab I'M VERY GRATEFUL!
Huh is there a vid you did on eDNA sequencing? To me thats bery interesting...how all that DNA is seperated and sequenced.
Thanks for your question. 🤓 No, I've not covered environmental DNA sequencing yet, but it is fascinating. A good idea to add to my list. 👍
@@ClevaLab I find it fascinating too. I read about how filters from weather monitoring stations can "vacuum" DNA out of the air. I would love to learn how to go build them and go from filters to looking for new species. Combined with water filtration it be a very interesting technique for looking for new sources in far flung remote locations.
Bravo.....ok......estract dna from urea.....cheers
Thanks for your comment. 🤓 Urea is a compound (a pure substance made of two or more elements chemically bound together). Do you mean urine? You can extract DNA from urine. There's protocols for this using commercial DNA extraction kits.
@@ClevaLab urine.....yes.......stem cells how to........
Can you do microarray?
Thank you very much
Thanks for your comment. 🤓 Good suggestion, I put it on my list!
Can you please explain what is the function of reading index at 5:04? Thank you so much i love you
yeah i have the same question with you 🥲
5:50 it’s like a tag for each sample
Thanks for your comment. 🤓 This index is the second index read when you use dual indexes. Using dual indexes instead of single indexes means you can multiplex more samples in the same flow cell lane. So, when you use a single indexed library, you can use up to 48 libraries per flow cell lane. In contrast, you can use up to 384 samples with dual-indexed libraries. Of course, the number of samples you'd want to multiplex will depend on the NGS application. Please let me know if you have further questions or if this wasn't quite what you were asking.
@ngockim6419 - Thanks for your comment. 🤓 I've answered this question on the thread. Please let me know if you have further questions.
How do you know which strand is the forward and which one is the reverse when the bridge is formed?
Thanks for your question. 🤓 Each library fragment has two adapters on each end. One end of the adapter contains the P7 sequence, and the other has the P5 sequence. On the flow cell, there are two types of oligos, one containing the P7 sequence and one containing the P5 sequence. It doesn't matter if the library is attached to the P7 or P5 oligo in the initial library attachment step. It only matters that a single library strand is bound far enough away from another strand that it doesn't overlap with the next cluster after cluster generation.
The strands bound to the P5 oligo are deemed to be the reverse strand. A sequence within the P5 oligo is either chemically or enzymatically cut to detach these strands from the flow cell surface.
The short answer is that you can tell the reverse strand because it's bound to the P5 oligo.
Coud you do an explanation for elementary/middle school students?
Thanks for your comment. 🤓 I'm currently aiming for university level and above, but it's a good idea. I'll put it on my list. 👍
@@ClevaLab I understand! Thanks!
What are first-index and second-index reads? (I understand what index means, but what are these and what signioficance do they have?) Could anyone clarify, please?
Thanks for your question. 🤓 Indexes are used to identify the sample. Libraries are prepared from samples separately, and a different index is used for each sample. Then, each of the libraries gets mixed into one tube before sequencing. The reads can be identified as belonging to a particular sample because the first index is read just before the sequencing read.
If single or dual index reads are used depends on the application. For gene expression where no sequence information is needed, only an ID of the gene, then a single index read is OK. But, if you use dual indexes, you will read both ends of your insert. This double reading is called paired-end sequencing. The first and second index reads identify both the sample and if it is the forward or reverse end of the insert. For large inserts aligned to the reference sequence, the bioinformatics software will know that the reads are from the same insert. Paired-end reads make it easier to align the reads to the reference.
I hope that answers your question. Please let me know if you have any further questions.
Am I right that since the moment of filtering and maping it is all done with software, not sequencer?
Thanks for your comment. 🤓 That's correct. Once the sequence is read by on the sequencer, the filtering, mapping, and further analysis are done with software on a computer.
Thanks. The Human reference DNA how was create?
Thanks for your comment. 🤓 The human reference genome was created by Sanger sequencing done by the Human Genome Project. I also have a video on Sanger sequencing here: th-cam.com/video/X9566yI2cBo/w-d-xo.html
@@ClevaLab thanks a lot :)
Bottom line for a layman:
1.) Do all of these complex procedures actually defeat Cancer?
2.) If so, what's the track record for eliminating different Cancers?
3.) Is NGS convincing enough in efficacy that Medicare will pay for it?
4.) If Medicare will not pay, what is the cost to an individual?
Thanks for your question. 🤓 NGS is used in cancer for personalized medicine and is still in its early days. The most common tests are NGS panels of genes, with anywhere from tens to hundreds of genes in one panel. These panels generally are for diagnostic tests for inherited cancers or testing of the tumour itself for specific mutations. NGS testing for specific mutations in the tumour is done to determine the best drug treatment for the patient. The drug treatment could be an existing treatment or a clinical trial.
There is evidence that NGS can help increase cancer patients' overall survival by targeting their therapy. See here: doi.org/10.1200/PO.22.00715
I assume you mean Medicare in America. Yes, FDA-approved NGS tests for the diagnosis and treatment of advanced cancers are approved by Medicare. Please see here: tinyurl.com/3rudp8cm
I'm not sure of the out-of-pocket costs for any NGS testing not covered by Medicare.
How much depth is needed for the microbiome sequencing??
Thanks for your question. 🤓 The depth needed for microbiome sequencing will depend on if you want to do 16S or Shotgun sequencing. Illumina has a good guide (Shotgun Metagenomics Methods Guide) you can find in this link 🔗 sapac.illumina.com/areas-of-interest/microbiology/microbial-sequencing-methods/microbial-whole-genome-sequencing.html.
For 16S, a 10,000x depth per sample is recommended. For Shotgun sequencing, 0.5 to 1 million reads for taxonomic profiling (i.e. what bacteria, fungi, and viruses are there) and 80 million reads for monitoring antimicrobial resistance (This info is directly from Illumina's "Shotgun Metagenomics Methods Guide").
@@ClevaLab thanks for this description of read depth! How do some companies (like illumina) confirm the specific read depth number assigned to workflows like 16s or Shotgun?
@@mosestinio537 Hi Moses, thanks for your question. 🤓Once the run is complete, the reads are filtered and demultiplexed. The resulting fastq file is then used in bioinformatics software like BaseSpace and the Metagenomics App. Finally, the bioinformatics software will report how many reads were obtained from each sample, along with a detailed sample analysis.
Does this answer your question? Please let me know if you have any further questions.
How do they isolate the forward from the reverse strand?
Thanks for your question. 🤓 The library is denatured using Sodium Hydroxide to separate the forward and reverse strands. The forward or reverse strand can bind to the oligos and get amplified by bridge amplification. However, the reverse strands are cut by an enzyme so that only the forward strands are present before sequencing starts. I hope this answers your question.
Please let me know if you have further questions.
@@ClevaLab One follow up question, how does the enzyme identify the reverse strand?
@@Scriabinfan593 The adapter contains a sequence that can be cleaved. Unfortunately, Illumina is not specific about how they do this. It must be proprietary. They only say that "the reverse strand is removed by specific base cleavage".
@@ClevaLab Thanks for your response!
What is the first index and second index???
Thanks for your question. 🤓 Two indexes are used to allow multiplexing of a higher number of samples. Multiplexing is mixing multiple sample libraries and running them in the same flow cell. You can read more about this here: sapac.illumina.com/techniques/sequencing/ngs-library-prep/multiplexing.html
❤😊
Thanks for commenting. 🤓 I'm glad you liked it.
You didn't tell properly how reverse strand not attaches,
Thanks for your question. 🤓 Sorry for the slow reply. That's a very good question! Good thinking. 👍I only show one binding in the interests of simplicity of animation. One strand of the library will bind to the flow cell, but it can bind to either of the oligos (p7 or p5). This is because both oligos are complementary to that strand. After clonal (bridge) amplification, the reverse strand is cut and washed away, so only the forward strands are left for sequencing.
huh? balikan ko ni na comment if naintindihan ko na
🤓 Did you understand?
Why the indexes are sequenced
Thanks for your question. 🤓 The indexes are sequenced to tell you the sample each sequence belongs to.
👀
Crazy a.f
This is so complicated