I am planning on performing an illumina multiplex sequencing run on a library of sequences that I have compiled. They are digested out of plasmid and consist of ~10 unvaried nucleotides, 60 varied nucleotides, ~40 unvaried nucleotides, 60 varied nucleotides, then 10 unvaried nucleotides in that order. I have seen in previous papers that there are some issues with the cluster identification step for sequences which are unvaried in their first few nucleotides. Is this still a major problem? Is there a way to get around this without adding extra varied nucleotides to the ends of my sequences? Or is this the only robust solution?
Seqanswers Leaderboard Ad
Collapse
Announcement
Collapse
No announcement yet.
X
-
You need to design a custom sequencing primer.
Use the sequence of the adapter+sequence of your non-variable region(10 bases)
Then create a sequencing primer that is complementary to the last base of your nonvariable region-then work back.
When the Tm of the primer is above 65 (that way it will work on both HiSeq and MiSeq) you have that primer synthesized and HPLC purified.
Use the primer at 100uM.
Whoever is doing you sequencing will know how to use it.
-
If you are sequencing a small number of plasmid-derived inserts, then you are likely to not need the full number of reads in a lane. An alternative to creating a custom primer is to just spike your library into a control lane of PhiX on a HiSeq, or mix in a higher-complexity library (usually someone has something they want a little more sequence for) on a MiSeq. It seems like the recent software changes have made the MiSeq less sensitive to complexity reductions anyway.Providing nextRAD genotyping and PacBio sequencing services. http://snpsaurus.com
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
22 views
0 likes
|
Last Post
by seqadmin
04-11-2024, 12:08 PM
|
||
Started by seqadmin, 04-10-2024, 10:19 PM
|
0 responses
24 views
0 likes
|
Last Post
by seqadmin
04-10-2024, 10:19 PM
|
||
Started by seqadmin, 04-10-2024, 09:21 AM
|
0 responses
19 views
0 likes
|
Last Post
by seqadmin
04-10-2024, 09:21 AM
|
||
Started by seqadmin, 04-04-2024, 09:00 AM
|
0 responses
51 views
0 likes
|
Last Post
by seqadmin
04-04-2024, 09:00 AM
|
Comment