Thansk austinso,
I think denaturation at a higher temperature sounds like a very good idea to tackle denaturation problems. (we did the standard NaOH denaturation 5min at RT, then mix with cold HT1).
Just to be safe: for the TruSeq RNA libraries stored as dsDNA, you suggest to mix with NaOH, 5min RT, ice, mix with cold HT1, 2min 95deg, ice, pool, load.
Woudl you suggest doing the NaOH before storage in later library preps as they suggest to do with the SGP plate?
Thanks, Mareen
Seqanswers Leaderboard Ad
Collapse
Announcement
Collapse
No announcement yet.
X
-
Originally posted by mareen_engel View PostThank you Phillip.
While I agree that we may sometimes tend to overrate the problem of duplicates in RNA-Seq, I think it's a slightly different thing here, where I do see 3 Mio duplicates exactly next to each other on the flowcell with overall duplicate numbers which in my eyes are not high enough to render this statistically possible? Usually we see some 0-low hundreds of those optical duplicates even in our low-complexity runs?
For me this sounds like a problem in library prep/denaturation/clustering, would you disagree?
Best, Mareen
Doing library denaturation with NaOH for 2 min at 96C as recommended for TruSeq libraries (http://support.illumina.com/content/...15027983-c.pdf, p.36) did the trick in removing these duplicates.
A.
Leave a comment:
-
Thank you Phillip.
While I agree that we may sometimes tend to overrate the problem of duplicates in RNA-Seq, I think it's a slightly different thing here, where I do see 3 Mio duplicates exactly next to each other on the flowcell with overall duplicate numbers which in my eyes are not high enough to render this statistically possible? Usually we see some 0-low hundreds of those optical duplicates even in our low-complexity runs?
For me this sounds like a problem in library prep/denaturation/clustering, would you disagree?
Best, Mareen
Leave a comment:
-
I don't know -- I don't really trust programs that claim to be able to find optical or PCR "duplicates". How do these programs know they are not just biological duplicates? RNA will have a highly non-normal distribution with the possibility of just a few transcripts making up a large percentage of the library. If there is any fragmentation/ligation bias in library construction then you will see lots of the same sequence popping up irrespective of any instrument/PCR duplication.
--
Phillip
Leave a comment:
-
High number of optical duplicates on MiSeq
Dear all,
we've run a Illumina TruSeq mRNA nonstranded library (mouse brain lower end of input recommendation) on a MiSeq for quality control with v3 Reagent Kit, 2*75 PE. The run has very good QCs, good cluster density and some 22 Mio reads.
After pairing the reads, Picard MarkDuplicates reports more then 3 Mio optical duplicates (i.e. duplicates less then 100pixel apart on the flow cell, similar results for 10pixel) next to roughly the same amount of "real" PCR duplicates (50% of QC20 reads after throwing the optical duplicates).
If analyzing the SE data without pairing, the number of optical duplicates is reduced to 0, thus it seems not to be a cluster-read failure.
For me, this indicates that there's 3 Mio clusters on the flowcell next to a cluster with their reverse complement strand which seems to be much above chance level even with a high PCR-duplicate RNA-Seq?
Potential explanations put forward by the representative contacted are incomplete library denaturation prior to loading (thus partially dsDNA library molecules hybridize to the flowcell and build two reverse complement clusters) or low complexity of the library (however, 3 Mio sounds really above chance level for me, the comeplexity of the library doesn't seem to be that bad?)
Has anybody ever seen this? Any ideas what is causing this high number of optical duplicates?
Or is this number of duplicates simply expected on a standard RNA-Seq on low input (~200ng)?
Any ideas or recommendations are very much appreciated!
Many thanks!
Regards,
Mareen
Edit1:
Ps.: Not sure which heading this thread should go to, please move if you have a better idea. Thanks!
Edit2:
library PCR was 12 cyclesLast edited by mareen_engel; 02-05-2015, 04:13 AM.Tags: None
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...-
Channel: Articles
04-22-2024, 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, Today, 11:49 AM
|
0 responses
13 views
0 likes
|
Last Post
by seqadmin
Today, 11:49 AM
|
||
Started by seqadmin, Yesterday, 08:47 AM
|
0 responses
16 views
0 likes
|
Last Post
by seqadmin
Yesterday, 08:47 AM
|
||
Started by seqadmin, 04-11-2024, 12:08 PM
|
0 responses
61 views
0 likes
|
Last Post
by seqadmin
04-11-2024, 12:08 PM
|
||
Started by seqadmin, 04-10-2024, 10:19 PM
|
0 responses
60 views
0 likes
|
Last Post
by seqadmin
04-10-2024, 10:19 PM
|
Leave a comment: