Has anyone tried low-diversity amplicon sequencing (16s) with the new Illumina recommendation of only a 5% spike of phiX? I have a couple of people who want to start prepping their libraries and are wondering if they need to artificially add diversity or not. Thanks!
Seqanswers Leaderboard Ad
Collapse
Announcement
Collapse
No announcement yet.
X
-
Depends on the complexity of your amplicon library.
From our own experiences, if you do not have a complex library you will need to add complex sequence to the 5' of your amplicon or use more phiXLast edited by JackieBadger; 06-26-2013, 06:37 PM.
Comment
-
Originally posted by JackieBadger View PostDepends on the complexity of your amplicon library.
From our own experiences, if you do not have a complex library you will need to add complex sequence to the 5' of your amplicon or use more phiX
Have you tested this with the recent release of the MiSeq software which addresses issues with sequencing amplicon libraries?
Comment
-
Originally posted by kmcarr View PostJackie,
Have you tested this with the recent release of the MiSeq software which addresses issues with sequencing amplicon libraries?
-Tom
Comment
-
Originally posted by thomasblomquist View PostIs this the release I've heard of where the RTA won't kill the entire run if it can't focus on a particular step, or where/when, if possible, the RTA will define colonies/clusters later if cluster differentiation is better at later flows of nucleotides?
-Tom
We have done a couple of runs thus far using the Caporaso & Knight primers. We had 16-18 M raw clusters, 13-15 M PF clusters, 93% ≥ Q30 on read 1, 87% ≥ Q30 on read 2 (PE 150 reads). We were shooting for 5% PhiX but ended up ~10%.
Comment
-
Originally posted by kmcarr View PostNot quite. Here are the Release Notes. Specifically it is the changes added in RTA v1.17.28 which make significant improvements to amplicon sequencing without need for low cluster densities, hard coded matrix and phasing values or very high (25-50%) PhiX. Recommendations for amplicon sequencing with this version of the software are standard cluster densities of 500-1200K/mm^2 and only 5% PhiX.
We have done a couple of runs thus far using the Caporaso & Knight primers. We had 16-18 M raw clusters, 13-15 M PF clusters, 93% ≥ Q30 on read 1, 87% ≥ Q30 on read 2 (PE 150 reads). We were shooting for 5% PhiX but ended up ~10%.
Regards,
-Tom
Comment
Latest Articles
Collapse
-
by seqadmin
The sequencing world is rapidly changing due to declining costs, enhanced accuracies, and the advent of newer, cutting-edge instruments. Equally important to these developments are improvements in sequencing analysis, a process that converts vast amounts of raw data into a comprehensible and meaningful form. This complex task requires expertise and the right analysis tools. In this article, we highlight the progress and innovation in sequencing analysis by reviewing several of the...-
Channel: Articles
Today, 07:48 AM -
-
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 -
ad_right_rmr
Collapse
News
Collapse
Topics | Statistics | Last Post | ||
---|---|---|---|---|
Started by seqadmin, Today, 07:17 AM
|
0 responses
11 views
0 likes
|
Last Post
by seqadmin
Today, 07:17 AM
|
||
Started by seqadmin, 05-02-2024, 08:06 AM
|
0 responses
19 views
0 likes
|
Last Post
by seqadmin
05-02-2024, 08:06 AM
|
||
Started by seqadmin, 04-30-2024, 12:17 PM
|
0 responses
20 views
0 likes
|
Last Post
by seqadmin
04-30-2024, 12:17 PM
|
||
Started by seqadmin, 04-29-2024, 10:49 AM
|
0 responses
28 views
0 likes
|
Last Post
by seqadmin
04-29-2024, 10:49 AM
|
Comment