Dr. Rob Edwards from San Diego State University describes how Illumina paired-end sequencing works.
Пікірлер: 61
@yyl53365 жыл бұрын
Thank you for uploading of this video. I finally understood what is P.E sequencing.
@julianstanley95165 жыл бұрын
Phenomenally well-thought-out teaching style. Thanks for taking the time to put these together!
@Artas19844 жыл бұрын
He makes a mistake at 3:48 - the fragmented DNA with adapters is washed away, and it's the new IMMOBILIZED A primer-extended DNA that should form the bridge with an immobilized B primer on the glass slide, and NOT the loose DNA...
@mikewheeler17123 жыл бұрын
yes! I noticed that too. i thought, whoa wait a minute.... you missed something.
@schlag_sensei2 жыл бұрын
Thanks ! I've just taken a look to the Illumina video, and you are right.
@Ismailkhan-mz2mz2 жыл бұрын
Plz send me definition of pair end sequencing
@Yetus2 жыл бұрын
I noticed this as well lol. I’m learning!
@danielsiqueiradeoliveira39294 жыл бұрын
Amazing presentation! Congratulations
@sung4613 жыл бұрын
This is the best explaination ever!
@jenniferalmon47935 жыл бұрын
Beautiful presentation!!
@amritabhattacharjee45963 жыл бұрын
The original template which hybridises with flowcell oligo gets washed off after compimentary strand synthesis and then this complimentary strand bends over to the nearby oligo for bridge formation and cluster generation.
@Ismailkhan-mz2mz2 жыл бұрын
Plz define pair end sequencing mam
@Karsten13984 жыл бұрын
Very well explained, thank you.
@nom3nnescio3 жыл бұрын
lot of errors, so n8t so great
@nirmalkumars79932 жыл бұрын
Excellent presentation 👍
@Rodrifuuu10 ай бұрын
Great explanation, thank you so much.
@ignasijs97343 жыл бұрын
Thanks for your video, as we ve got reads 1 and 2. Are the reads 1 the forward and the reads 2 the reverse.
@mathieujoos63835 жыл бұрын
Isn't there a mistake in the picture around 4.15? The DNA attached to the glas slide will fold back while the other DNA strand is gone by denaturation or am i wrong?
@karanthakar6554 жыл бұрын
U are right.. there are a lot of misnomers. Including one at 05:07 where he shows synthesis in both direction as if DNA pol can go both 5'->3' and reverse.. Synthesis occurs on 2 different sequences and not on the same one..
@sublimetrance4 жыл бұрын
Yeah this was confusing me at first as well. Really makes you wonder if the instructor really fully understands this material.
@santamsaha94154 жыл бұрын
@@karanthakar655 exactly... Even I was moved by this fact
@User9527_2 жыл бұрын
You are right and he's wrong.
@nic59583 жыл бұрын
I've watched a lot of illumina sequencing overviews and this one is the best
@changfengchen92503 жыл бұрын
Such a great explanation, thank you!
@duhyadiolivagarcia15035 жыл бұрын
Great video
@balakumara71402 жыл бұрын
Thank you sir for the information
@GSurafel5 жыл бұрын
Thank you very much for this incredibly helpful video. I'm not sure if you will read this or if you take requests, but I would appreciate your take on chromosomal walking and CRISPR systems. Respectfully, Surafel G.
@salmalegdali82315 жыл бұрын
sir, thank you (casablanca/morocco)
@bhaveshohal33903 жыл бұрын
Nice... Understood the concept ☺☺
@marybelarenas644 жыл бұрын
First of all great video. Second of all, one quick question regarding moment 7:26, wouldn't a gap in the sequence be bad? seeing as we want to KNOW the bp of that piece of DNA, why would we only sequence the ends? I hope that question made a bit of sense hahaha , thank you!
@redseaflora8497 Жыл бұрын
I have the same question, but my understanding is that even if you are not sequencing that middle bit, just knowing the length of that long fragment and the regions that flank it lets you figure the length of the repetitive regions in the middle that would otherwise muck up assembly. You are basically using the longer mate pairs to help put shorter reads in context. That is as I understand it, if anyone has anything else to add please feel free.
@ryandikdan4 жыл бұрын
Is he writing backwards?
@winproduction7585 Жыл бұрын
Thank you sir
@nicolelaurent4128 Жыл бұрын
Excellent teaching. Thank you.
@deepakkumarverma46595 жыл бұрын
Great sir please make another new videos
@londonwallace6460 Жыл бұрын
Did this brilliant guy learn to write in a mirror? Or am I missing something. Well done. Thank you.
@RiQ9ZP0NxB11 ай бұрын
The video has been flipped.. ;) I'm pretty sure he's right-handed.
@emfowler295 жыл бұрын
Excellent resource, use of sequencing to detect structural chromosome rearrangements would be great!
@rebeccaeliscu34605 жыл бұрын
I don't fully understand how a long fragment (3000 bp example) helps with mapping repetitive RNA reads. Could you elaborate on this?
@erpampa945 жыл бұрын
I'll try to be helpful even though I'm not an expert, in the human genome for example there are many ripetitive regions and this could be a problem if you do a single-end sequencing, because you could have a sequenced read that is complementary to these repetitive regions and you can't align it with a high degree of mapping quality (means that you could place it at multiple position in the genome). If you do a paired-end sequencing as long as both reads don't fall into a repetitive structure you can anchor one with certainty even if the other doesn't anchor really precisely.
@nicholastan70075 жыл бұрын
I don't understand 5:25. Why is it possible to sequence the same DNA fragment from both end ? I thought sequencing can only happen in the 5' to 3' direction. Can anyone please help me ? Also, are the strands, double stranded ?
@srgk265 жыл бұрын
Cos the reverse strand is not sequenced directly as it is, in 3'-->5'. It goes through another step to synthesise 5'-->3'. This video is clearer onto the actual protocol: kzbin.info/www/bejne/fn7cdKSNndx1bqc
@mirelamatecic81725 жыл бұрын
Well actually, both oligos (pink and blue) were supposed to be attached with their 5 prime ends to the solid surface, so the illustration of the paired primer (oligo) extension is confusing, since the drawing implicates that template has only 3 prime ends. Yup, something not right.
@erikmuller62722 ай бұрын
7:00 150bp from both end will have 100 bp overlap on 200bp . Like total length 12 bp has 6/6 no overlap ,but 12 shorten to 8bp will have 4 overlap of 6/6 both end read
@macksoneh4 жыл бұрын
6:55 For a 200 bp fragment, the overlap would be 100bp.
@mathisventura36863 ай бұрын
I’m a little bit lost in my analysis: - I am expecting a 70bp amplicon at the end. - When I look at the size of my reads 1 and 2 before reassembling, I have 12 to 131 bp for R1, and 21 to 148 bp for R2. - How, with these highly distributed read lengths, am I supposed to choose the size for reassembling?
@nikhilshinde45224 жыл бұрын
Sir... will you please provide name or link for reference notes
@yashan.b.8346 Жыл бұрын
If my fragment size is 500bp, and read length is 150*2 with 300 cycles, I get inner overlap of 100bp. Does this overlap effect my overall depth of coverage/sequencing results?
@tomgag3552 Жыл бұрын
The inner overlap of 100bp that you are getting with 150bp paired-end reads will not affect your overall depth of coverage or sequencing results significantly. In paired-end sequencing, the inner overlap is generated when the two reads from opposite ends of the same DNA fragment overlap with each other. This overlap helps to increase the accuracy of the sequencing by allowing errors to be corrected and gaps to be filled. However, the impact of this overlap on the overall depth of coverage depends on various factors such as the size and complexity of the genome or transcriptome being sequenced, the sequencing platform used, the sequencing depth, and the bioinformatics pipeline used for data analysis. In general, the depth of coverage is determined by the number of reads that align to a specific region of the genome or transcriptome. The higher the number of reads, the higher the depth of coverage, which leads to better accuracy and sensitivity in detecting genetic variations or gene expression levels. Therefore, if the sequencing platform and the bioinformatics pipeline are optimized to handle paired-end reads with inner overlaps, the impact of the overlap on the depth of coverage should be minimal, and you should still get reliable sequencing results
@tabea28413 жыл бұрын
I don't really get it.. Why would we need to sequence from both ends? Can't we just sequence the full fragment in one direction? Isn't that what "normal" Illumina sequencing is doing? what's the advantage of the paired-end approach? Thanks for an answer in advance! :)
@nom3nnescio3 жыл бұрын
you can get over repetetive areas
@CrazycruxGaming3 жыл бұрын
are...are you writing backwards?
@huynamnguyen71744 жыл бұрын
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? Thank you.
@Hoffenheim95 Жыл бұрын
are you still waiting for an answer?
@fedouaoumamar36585 жыл бұрын
thank you but where is the rest i mean how to sequence dna after these steps!!!
@gandhimusic7812 жыл бұрын
As great as this video is made, there are several core-errors with this explanation.
@Biocreator3 жыл бұрын
pls remove and remake with corrections!
@sam2theammyk93 жыл бұрын
Its clear he doesn't know what is A' and B'. It isn't as intuitive as one might think. That is why he stopped labeling
@andreaisonline2 жыл бұрын
Good luck using a 3kb library size with an Illumina device…