Ion Torrent Sequencing

Dr. Rob Edwards from San Diego State University outlines Ion Torrent Sequencing.
For an overview of genome sequencing, see the course manual: linsalrob.gith...

[@sirusThu]

Great content, please keep it up :)

[@duyeninh8915]

wow, reverse writing. Thank you for the clear explanation, Dr. Edwards.

[@pjmvdbroek]

it is a software tool/smart board

[@xruan6582]

excellent, as always

[@duyeninh8915]

Where can I find the rest of your lecture?

[@maxou2175]

Thank you so much ! One question : You just add one dNTP after the other ? Like how do you make the difference between adding a dATP from adding a dCTP ? Why don't the pH decrease like adding several dATP as you explained ? Thank you

[@conclusionforeign8568]

let's say we're sequencing, and our remaining template sequence is
3'-ACCCCATG-5'
5'->
1. buffer with dATP flows through IonTorrent machine, polymerase rejects it since it's not complementary to A
3'-ACCCCATG-5'
5'->
2. buffer is washed away
3. buffer with dTTP flows through, T is incorporated and pyrophosphate molecule is released
3'-ACCCCATG-5'
5'-T->
4. change in pH is noted, buffer is washed away
5. buffer with dCTP flows through, polymerase rejects it as it's not complementary to C
3'-ACCCCATG-5'
5'-T->
6. buffer is washed away
7. buffer with dGTP flows through, 4 Gs are incorporated
3'-ACCCCATG-5'
5'-TGGGG->
8. a bigger change in pH is noted but it's hard to tell exactly how many Gs were incorporated. 3? 4?
9. we cycled through buffers with ACT and G, so we begin the cycle again. Buffer with dATP flows through
3'-ACCCCATG-5'
5'-TGGGG->
10. buffer is washed awas, buffer with dTTP flows through
3'-ACCCCATG-5'
5'-TGGGGT->
11. wash, and then dCTP
3'-ACCCCATG-5'
5'-TGGGGT->
12. wash and dGTP
3'-ACCCCATG-5'
5'-TGGGGT->
13. wash and dATP
3'-ACCCCATG-5'
5'-TGGGGTA->
14. wash and dTTP
3'-ACCCCATG-5'
5'-TGGGGTA->
15. wash and dCTP
3'-ACCCCATG-5'
5'-TGGGGTAC->
16. we're done!