illumina paired end sequencing

Dr. Rob Edwards from San Diego State University describes how Illumina paired-end sequencing works.

[Artas]

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...

[amritabhattacharjee]

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.

[julianstanley]

Phenomenally well-thought-out teaching style. Thanks for taking the time to put these together!

[yyl]

Thank you for uploading of this video. I finally understood what is P.E sequencing.

[mathieujoos]

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?

[nicholastan]

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 ?

[londonwallace]

Did this brilliant guy learn to write in a mirror? Or am I missing something. Well done. Thank you.

[macksoneh]

6:55 For a 200 bp fragment, the overlap would be 100bp.

[mathisventura]

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?

[erikmuller]

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

[nic]

I've watched a lot of illumina sequencing overviews and this one is the best

[GSurafel]

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.

[ignasijs]

Thanks for your video, as we ve got reads 1 and 2. Are the reads 1 the forward and the reads 2 the reverse.

[Rodrifuuu]

Great explanation, thank you so much.

[marybelarenas]

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!

[rebeccaeliscu]

I don't fully understand how a long fragment (3000 bp example) helps with mapping repetitive RNA reads. Could you elaborate on this?

[loveforbioinfo]

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?

[tabea]

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! :)

[nikhilshinde]

Sir... will you please provide name or link for reference notes

[emfowler]

Excellent resource, use of sequencing to detect structural chromosome rearrangements would be great!