Tweet
SOLiD PE Chemistry
Hello, we've been trying out the PE 50/35 sequencing chemistry and the reverse (35bp) read quality is poor. Does anyone know which step(s) of the 35bp chemistry makes it less efficient than the forward reaction? Is it due to the 5'->3' ligase or an aspect of the extra capping/dephosphorylation steps?
-
-
My guess is that it is the latter.
But another factor is that Lifetech has not been tinkering with the chemistry they use to do 5' extensions as they have the 3' extensions. I hope this means there will be improvement as that chemistry is optimized. Currently, when all goes well, the 5' extension chemistry looks to me about like things did under v2 3' chemistry.
--
Phillip -
I've also seen poorer quality on the reverse read.
Despite a few people trying to educate me, I'm still confused why the reverse reads are more difficult. But, it's very hard to see how the ligation step could be the issue -- ligase shouldn't know which fragment you think is front and which you think is back, it's just looking to make a join between two fragments.Comment
-
Proteins are often described as the workhorses of the cell, and identifying their sequences is key to understanding their role in biological processes and disease. Currently, the most common technique used to determine protein sequences is mass spectrometry. While still a valuable tool, mass spectrometry faces several limitations and requires a highly experienced scientist familiar with the equipment to operate it. Additionally, other proteomic methods, like affinity assays, are constrained...04-04-2024, 04:25 PM -
Despite advancements in sequencing platforms and related sample preparation technologies, certain sample types continue to present significant challenges that can compromise sequencing results. Pedro Echave, Senior Manager of the Global Business Segment at Revvity, explained that the success of a sequencing experiment ultimately depends on the amount and integrity of the nucleic acid template (RNA or DNA) obtained from a sample. “The better the quality of the nucleic acid isolated...03-22-2024, 06:39 AM
Comment