Hi there, does anyone know if there is a maximum size fragment that won't be sequenced by a MiSeq? The approach is paired end 250x250 on a size selected library of fragments. I'd like to use size selection to sequence a 500bp range of fragments (i.e. 900-1400 or 1200-1700 or 1500-2000), but have heard that fragments over ~1200 don't get sequenced. Is this assertion true? or can you sequence fragments over 1200 bases?
Seqanswers Leaderboard Ad
Collapse
Announcement
Collapse
No announcement yet.
X
-
Larger fragments don't hybridize to the MiSeq flow cell as well as smaller ones. This can be seen when you have a small amount of adapter dimer in your (larger) libraries. The adapter dimer binds preferentially and what started out as a small percentage of adapter dimer ends up being half your sequencing data! However, the main problem with large fragments is that they tend to "flop over" into other clusters when they're bridge-amplifying so the MiSeq can't differentiate between clusters. Why do you want such large fragments?
-
Thank you - yes, I'm familiar with the bias towards smaller fragments, but am hoping to identify if there is an upper limit.
The reason for want to sequence larger fragments is that for targeted sequencing of the HLA region the further the distance between the paired reads, the greater the ability to phase the variants present between the two alleles in the sample.
Do you know if there is an upper empirical limit on what can be successfully sequenced on the MiSeq?
Comment
-
Comment
-
Following paper has described sequencing large amplicons on MiSeq:
http://biorxiv.org/content/early/2014/11/06/010967
Materials and Methods:
http://biorxiv.org/content/biorxiv/s...1/010967-1.pdf
Comment
-
Hi Peter,
To a first approximation, the issue is only one of competition from shorter amplicons. If you can completely rid yourself of all shorter amplicons, then the longer ones can cluster.
Generally everyone wants to blame the failure of the long amplicons to sequence on their supposedly creating large diffuse clusters. But I think this is a minor issue because I didn't see a dramatic change in the diameter of clusters between 500 and 1000 bp amplicons. (See the thread linked to by GenoMax above.)
--
Phillip
Comment
Latest Articles
Collapse
-
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 -
-
by seqadmin
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...-
Channel: Articles
03-22-2024, 06:39 AM -
ad_right_rmr
Collapse
News
Collapse
Topics | Statistics | Last Post | ||
---|---|---|---|---|
Started by seqadmin, 04-11-2024, 12:08 PM
|
0 responses
26 views
0 likes
|
Last Post
by seqadmin
04-11-2024, 12:08 PM
|
||
Started by seqadmin, 04-10-2024, 10:19 PM
|
0 responses
29 views
0 likes
|
Last Post
by seqadmin
04-10-2024, 10:19 PM
|
||
Started by seqadmin, 04-10-2024, 09:21 AM
|
0 responses
25 views
0 likes
|
Last Post
by seqadmin
04-10-2024, 09:21 AM
|
||
Started by seqadmin, 04-04-2024, 09:00 AM
|
0 responses
52 views
0 likes
|
Last Post
by seqadmin
04-04-2024, 09:00 AM
|
Comment