Hi, I am quite new to sequencing, but have been doing it the past 5 months with rather good results. I am wondering if anyone have sequenced beads significantly outside of Roches recommended range, i.e. 5-15%? I have lots of beads at 30% and would like to try...Is it likely not to work? What can happen? Would be grateful for any reply! Regards Eva
Seqanswers Leaderboard Ad
Collapse
Announcement
Collapse
No announcement yet.
X
-
I've sequenced beads with as high as 60% enrichment, but nothing over 20% has ever worked well here. I know of some users that won't sequence anything more than 10%. You'll probably end up with a high percentage of mixed and short quality reads, but should get some useable sequence as well, if you do end up sequencing those.
Good luck!
-
Originally posted by Anthony.287 View PostI've sequenced beads with as high as 60% enrichment, but nothing over 20% has ever worked well here. I know of some users that won't sequence anything more than 10%. You'll probably end up with a high percentage of mixed and short quality reads, but should get some useable sequence as well, if you do end up sequencing those.
Good luck!
Comment
-
The max we ever used was like 27% or so because we are in a hurry. The results were not bad and consistent with what we had obtained before for the same library with lower enrichment.
Other than that as a rule, we don´t sequence stuff above 20%
We do sequence stuff with more than 10% all the time, and we don´t notice bad results associated to that.
Comment
-
Originally posted by ChristianBourne View PostYou also have to consider that Roche are currently saying there may be an issue with 'unbound' DNA capture beads being carried through the enrichment and giving false enrichment values, this may also contribute to low numbers on the 454.
Also, how did you hear this? Roche has not come out and said this publicly, as far as I can tell.
Comment
-
Originally posted by bradfish View PostCan you clarify? By unbound, do you mean null beads, as in no DNA on them?
Also, how did you hear this? Roche has not come out and said this publicly, as far as I can tell.
Comment
-
Hi bradfish,
You betcha I can clarify baby! ;-)
Yes I mean 'null' beads. Our FSA told me personally. The number of washes in the protocol when enriching on the MPC may be unrealistic. But you know as well as I do that each sample can behave differently, and that's even before you factor in the differences in Lot numbers of kits!
It's yet another case of Roche not putting out a release of some kind informing all it's customers there may be an issue. I have spoken to fellow users who are not aware of any issues relating to inconsistencies with kits over the past few years or so. We all torture ourselves when we do everything correctly and our samples pass all the QC steps (including the new adaptor QC and post emPCR QC), when in fact it could be nothing we're doing.
Yes I know samples misbehave and don't work for a myriad of reasons, but cooperation from the company must be more forthcoming.
For example, I have had a recent order for 16 Flx+ runs. We have two Flx+ 454s. From four emulsion cups I enriched enough beads for four separate runs. I aloquotted my enriched beads out so all eight halves across my four runs contained exactly (sic) the same beads but I used two separate Lot numbers of reagents.
I saw a ~10% difference between machines and ~20% difference between Lot numbers.
Go figure. So factor that in with possible DNA capture bead manufacturing issues etc, and it adds up to a whole world of stress for us.
One thing you must do is check the wind direction and that you have your 'lucky' socks on when setting up a run. Now that's NGS science for you :-)Last edited by ChristianBourne; 07-20-2012, 01:41 AM.
Comment
-
Viva la revolution my friend!
I've got three years of stress, pain and misery built up thanks to Roche and their blooming 454. I used to be soooo happy running my GAiixs. Or am I just imagining that? The HiSeq and MiSeq I have work well enough, and there's no emulsion pcr
Maybe we should admit defeat and turn all the Flx+s back to titaniums.
Everyone is ooohing and aaaaahing over the 2x250 MiSeq, but come on it wouldn't touch a full 454 run for throughput. But the cost....Last edited by ChristianBourne; 07-20-2012, 01:34 AM.
Comment
Latest Articles
Collapse
-
by seqadmin
The field of epigenetics has traditionally concentrated more on DNA and how changes like methylation and phosphorylation of histones impact gene expression and regulation. However, our increased understanding of RNA modifications and their importance in cellular processes has led to a rise in epitranscriptomics research. “Epitranscriptomics brings together the concepts of epigenetics and gene expression,” explained Adrien Leger, PhD, Principal Research Scientist on Modified Bases...-
Channel: Articles
Yesterday, 07:01 AM -
-
by seqadmin
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...-
Channel: Articles
04-04-2024, 04:25 PM -
ad_right_rmr
Collapse
News
Collapse
Topics | Statistics | Last Post | ||
---|---|---|---|---|
Started by seqadmin, 04-11-2024, 12:08 PM
|
0 responses
39 views
0 likes
|
Last Post
by seqadmin
04-11-2024, 12:08 PM
|
||
Started by seqadmin, 04-10-2024, 10:19 PM
|
0 responses
41 views
0 likes
|
Last Post
by seqadmin
04-10-2024, 10:19 PM
|
||
Started by seqadmin, 04-10-2024, 09:21 AM
|
0 responses
35 views
0 likes
|
Last Post
by seqadmin
04-10-2024, 09:21 AM
|
||
Started by seqadmin, 04-04-2024, 09:00 AM
|
0 responses
55 views
0 likes
|
Last Post
by seqadmin
04-04-2024, 09:00 AM
|
Comment