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6.802/6.874/20.390/20.490/HST.506 Lecture April 2 2020 - Shared screen with speaker view
Seung-Hyun B Ko
56:37
Is barcode addition sequential?
xinxu
56:37
how can the barcode enter the cell and anneal to every mRNA?
xinxu
57:43
Thank you
Doris Fu
01:00:48
Is there a way to put UMI in the pool approach?
Meghana Kamineni
01:04:47
How does the template switch oligo limit the number of copies made?
Corban Swain
01:12:10
@Meghana which technology was your question referencing?
Meghana Kamineni
01:13:11
I was referring to the method described to deal with PCR bias where you limit the number of times you can amplify a template sequence?
xinxu
01:16:20
is multiplexing the same as sequencing different samples individually
xinxu
01:16:53
is this cheaper?
xinxu
01:17:47
thank you!
Corban Swain
01:21:20
@Meghana Using the template switch oligo is what enables the reverse transcription product to receive a unique molecular identifier (UMI). This UMI will enable you to determine if a PCR product was over amplified in your sequencing library. Because you have UMIs on each read, you can adjust the transcript abundance to be independent of the library abundance and better reflect the abundance in the cells.
Meghana Kamineni
01:22:20
That makes sense! Thank you!
Swati Kataria
01:26:32
What % of RNA is actually captured on individual beads that go on to be amplified and sequenced?
Swati Kataria
01:28:06
Yes
Swati Kataria
01:28:08
Thank you!