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...
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.
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?
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..
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 ?
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: ua-cam.com/video/HMyCqWhwB8E/v-deo.html
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.
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
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!
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.
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.
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?
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.
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?
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
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! :)
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.
Phenomenally well-thought-out teaching style. Thanks for taking the time to put these together!
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...
yes! I noticed that too. i thought, whoa wait a minute.... you missed something.
Thanks ! I've just taken a look to the Illumina video, and you are right.
Plz send me definition of pair end sequencing
I noticed this as well lol. I’m learning!
Thank you for uploading of this video. I finally understood what is P.E sequencing.
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.
Plz define pair end sequencing mam
This is the best explaination ever!
Amazing presentation! Congratulations
Beautiful presentation!!
Excellent presentation 👍
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?
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..
Yeah this was confusing me at first as well. Really makes you wonder if the instructor really fully understands this material.
@@karanthakar655 exactly... Even I was moved by this fact
You are right and he's wrong.
Very well explained, thank you.
lot of errors, so n8t so great
Great explanation, thank you so much.
Great video
Thank you sir for the information
I've watched a lot of illumina sequencing overviews and this one is the best
Such a great explanation, thank you!
Nice... Understood the concept ☺☺
Is he writing backwards?
Thank you sir
Thanks for your video, as we ve got reads 1 and 2. Are the reads 1 the forward and the reads 2 the reverse.
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 ?
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:
ua-cam.com/video/HMyCqWhwB8E/v-deo.html
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.
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
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!
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.
sir, thank you (casablanca/morocco)
Excellent teaching. Thank you.
Did this brilliant guy learn to write in a mirror? Or am I missing something. Well done. Thank you.
The video has been flipped.. ;) I'm pretty sure he's right-handed.
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.
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?
Great sir please make another new videos
6:55 For a 200 bp fragment, the overlap would be 100bp.
I don't fully understand how a long fragment (3000 bp example) helps with mapping repetitive RNA reads. Could you elaborate on this?
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.
are...are you writing backwards?
Sir... will you please provide name or link for reference notes
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?
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
Excellent resource, use of sequencing to detect structural chromosome rearrangements would be great!
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! :)
you can get over repetetive areas
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.
are you still waiting for an answer?
thank you but where is the rest i mean how to sequence dna after these steps!!!
pls remove and remake with corrections!
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
As great as this video is made, there are several core-errors with this explanation.
Good luck using a 3kb library size with an Illumina device…
This is wrong on so many levels don't watch this
for example?