Originally posted by Old guy
View Post
Seqanswers Leaderboard Ad
Collapse
Announcement
Collapse
No announcement yet.
X
-
-
Originally posted by beaver View PostHas somebody tried to systematically increase the amount of input DNA used for Rapid library nebulization (from the recommended 500 ng) to see how this affects the library yield? If you are doubtful you have an accurate quantification could it be harmful to add some more DNA to be on the safe side? If you are working with chromosomal DNA the amount is normally not limiting.
--
Phillip
Comment
-
rapid library problem
Hi,
We did 7 sequence runs using rapid libraries and only 2 turned out OK. The rest five have not produced long fragments reads. When I look the fragments size distribution, I initially thought, Oh, too much short fragments. But actually the distribution of short fragments were about the same in failed and successful runs. Its just in failed runs there were no long fragments. All my libraries (failed and good) average length is within the spec.
The other problem which was addressed here is that normally the average number of raw well we have is about 1.9M. But for rapid libraries - the average number of raw well is 1.3M. Does anybody experience similar problem?
I'm wondering, if short fragments issues is common for rapid libraries? Should we stop using rapid kit until Roche fix this problem?
To answer previously posted question, yes I accidentally did titration of rapid library without denaturation and I've got good results. Then I tried to use obtained cpb number and divided it by two for LV emPCr, and I received two times less beads than expected. I think you can't skip denaturation step, because you will end up with too many mixed data.
Comment
-
We had problems with short-read runs and Roche eventually agreed to replace our kits. Details here http://pathogenomics.bham.ac.uk/blog...are-not-alone/
Strongly suggest you report any problems you are having like this to the technical support team at Roche.
Comment
-
Thanks nickloman. We don't have problems with general libraries and amplicons. We only have problems with rapid libraries. In order to rule out the sequencing reagents, we tried to use two different lot numbers with the same DNA beads. We received similar results. The problem is most likely in library and less in emPCR step. But what is this problem?
Of course, I reported about our failed runs to Roche about 2 weeks ago, no response. I called again, no resolution. Seems that they don't have any ideas as well. That's why I went to this forum with question: is short reads and insufficient number of raw well is common for rapid libraries?
Comment
-
I put the sff from my rapid library run in geneious 5 and the read length is what I expect from my bio-analyzer results. Also if I check some wells from the run I can not see the B adaptor sequence so I believe I used the maximum read length.
If I however look at the read lengt distribution in the Roche RunBrowser the read lengths are 200 bp shorter because of the trimfilters. So I would recommend running the signalprocessing again with different filter settings if you want to stick to the Roche software.
Comment
-
Rapid Library Fragment Lengths
Originally posted by Cambridge454 View PostI am also having problems with the Rapid Library producing really long average fragment lengths >900bp. I use the recommended nebulization settings, which worked perfectly well for old version Shotgun libraries (600bp avg.). I have used the new vented caps and the rubber stopper/filter nebulizer set-up with very similar results. Should I increase the nitrogen pressure to say 2.5 bar instead of 2.1 bar?
Comment
-
Originally posted by Reshma View PostHi Cambridge454, how did you resolve this issue? What settings are you currently using for rapid library preps? My averages are above 900bp consistently! Any advice you can provide would be greatly appreciated. Can anyone else help me?
Comment
-
Originally posted by spet2522 View PostThe first post on this thread for a while, but is there any chance someone has a copy of the Rapid Library protocol that they could share?
Comment
Latest Articles
Collapse
-
by seqadmin
Next-generation sequencing (NGS) and quantitative polymerase chain reaction (qPCR) are essential techniques for investigating the genome, transcriptome, and epigenome. In many cases, choosing the appropriate technique is straightforward, but in others, it can be more challenging to determine the most effective option. A simple distinction is that smaller, more focused projects are typically better suited for qPCR, while larger, more complex datasets benefit from NGS. However,...-
Channel: Articles
10-18-2024, 07:11 AM -
-
by seqadmin
Non-coding RNAs (ncRNAs) do not code for proteins but play important roles in numerous cellular processes including gene silencing, developmental pathways, and more. There are numerous types including microRNA (miRNA), long ncRNA (lncRNA), circular RNA (circRNA), and more. In this article, we discuss innovative ncRNA research and explore recent technological advancements that improve the study of ncRNAs.
Nobel Prize for MicroRNA Discovery
This week,...-
Channel: Articles
10-07-2024, 08:07 AM -
ad_right_rmr
Collapse
News
Collapse
Topics | Statistics | Last Post | ||
---|---|---|---|---|
Started by seqadmin, 11-01-2024, 06:09 AM
|
0 responses
12 views
0 likes
|
Last Post
by seqadmin
11-01-2024, 06:09 AM
|
||
New Model Aims to Explain Polygenic Diseases by Connecting Genomic Mutations and Regulatory Networks
by seqadmin
Started by seqadmin, 10-30-2024, 05:31 AM
|
0 responses
14 views
0 likes
|
Last Post
by seqadmin
10-30-2024, 05:31 AM
|
||
Started by seqadmin, 10-24-2024, 06:58 AM
|
0 responses
24 views
0 likes
|
Last Post
by seqadmin
10-24-2024, 06:58 AM
|
||
New AI Model Designs Synthetic DNA Switches for Targeted Gene Expression in Specific Cell Types
by seqadmin
Started by seqadmin, 10-23-2024, 08:43 AM
|
0 responses
52 views
0 likes
|
Last Post
by seqadmin
10-23-2024, 08:43 AM
|
Comment