I have the same problem here
Seqanswers Leaderboard Ad
Collapse
Announcement
Collapse
No announcement yet.
X
-
I just had a similar "Object reference" failure about 185bp into read 1. In our case, cluster density was very low (Nextera XT normalised libraries pooled and diluted as per instructions but according to user, library concentration prior to going into normalisation step sounds low - 1-5ng/ul compared to 20-30ng/ul with libraries I've prepared myself).
Density was only 133K/mm2 which is way lower than our worst run to date (min 700k/mm2). We typically run 900-1300k/mm2.
Thanks for the tip on getting the MiSeq to analyse the data from the failed run, though - user wants to have a look at it although without the index reads I suspect it will be fairly meaningless.
Comment
-
Did you try to restart the run using the same cell and reagent set? It worked several times, I just used shorter run times and even got paired read completed. The reads have a lot of Ns and low quality regions, but with aggressive cleaning substantial set of useful data can be recovered. My feeling is sometimes BioAnalyzer traces are misleading in regard to average library size as contamination with shorter fragments can be significant yet not clearly visible. That results in a large portion of clusters being shorter then anticipated and vertical focus unexpectedly fails hitting the floor. That is usually signified by steep declines in the signal intensities on the graphs.
Comment
-
Originally posted by yaximik View PostDid you try to restart the run using the same cell and reagent set? It worked several times, I just used shorter run times and even got paired read completed. The reads have a lot of Ns and low quality regions, but with aggressive cleaning substantial set of useful data can be recovered. My feeling is sometimes BioAnalyzer traces are misleading in regard to average library size as contamination with shorter fragments can be significant yet not clearly visible. That results in a large portion of clusters being shorter then anticipated and vertical focus unexpectedly fails hitting the floor. That is usually signified by steep declines in the signal intensities on the graphs.
Comment
-
Here is my earlier post how to do this.
Bridged amplification & clustering followed by sequencing by synthesis. (Genome Analyzer / HiSeq / MiSeq)
If the run has aborted during read 1, then reagents are OK. However, if run terminated normally, either SE or PE, then reagents cannot be reused as MiSeq spits back something (backflush?) and ruins remaining reagents. What a waste!Last edited by yaximik; 03-18-2013, 07:15 AM.
Comment
Latest Articles
Collapse
-
by seqadmin
Technological advances have led to drastic improvements in the field of precision medicine, enabling more personalized approaches to treatment. This article explores four leading groups that are overcoming many of the challenges of genomic profiling and precision medicine through their innovative platforms and technologies.
Somatic Genomics
“We have such a tremendous amount of genetic diversity that exists within each of us, and not just between us as individuals,”...-
Channel: Articles
05-24-2024, 01:16 PM -
ad_right_rmr
Collapse
News
Collapse
Topics | Statistics | Last Post | ||
---|---|---|---|---|
Started by seqadmin, 06-03-2024, 06:55 AM
|
0 responses
12 views
0 likes
|
Last Post
by seqadmin
06-03-2024, 06:55 AM
|
||
Started by seqadmin, 05-30-2024, 03:16 PM
|
0 responses
26 views
0 likes
|
Last Post
by seqadmin
05-30-2024, 03:16 PM
|
||
Comprehensive Sequencing of Great Ape Sex Chromosomes Yields Insights into Evolution and Genetic Variability
by seqadmin
Started by seqadmin, 05-29-2024, 01:32 PM
|
0 responses
29 views
0 likes
|
Last Post
by seqadmin
05-29-2024, 01:32 PM
|
||
Started by seqadmin, 05-24-2024, 07:15 AM
|
0 responses
216 views
0 likes
|
Last Post
by seqadmin
05-24-2024, 07:15 AM
|
Comment