Hello. I am using Nextera XT to do whole genome sequencing of bacteria grown in stress conditions/high concentrations of antibiotics. What I found is, even if I follow the protocol correctly, I end up with a lowi(ish) cluster density (about 140K/mm2) and also The PF and Q30 reads are about 70%. I wonder what i coudl do to improve both cluster density and reads quality. If you have any tips please shout!
Seqanswers Leaderboard Ad
Collapse
Announcement
Collapse
No announcement yet.
X
-
From your post I can't be sure, but it sounds like you're sequencing your Nextera libraries on a NextSeq. If your loading concentration is resulting in 140K/mm2 densities, you can start by targeting a higher density (generally aim for 200K/mm2) with the formula [your starting concentration] x (target density)/(observed density) to get a new concentration to start with. I.E. if I loaded a pool at 1.7pM and observed 150K/mm2, I would multiply 1.7*(200/150) = 2.27pMwould be my new target loading concentration. To play it safe you could target the low end of the recommended density range (180K/mm2) but that is your choice.
As far as read quality, increasing the density without overclustering should help. The other obvious improvement would be spiking in a higher percentage of an ATCG balanced genome library will also help with basecalling. Illumina sells non-indexed PhiX to serve this purpose, but spiking in something easily separated from your bacterial samples might be a good idea.
-
Originally posted by thiNGS View PostHello. I am using Nextera XT to do whole genome sequencing of bacteria grown in stress conditions/high concentrations of antibiotics. What I found is, even if I follow the protocol correctly, I end up with a lowi(ish) cluster density (about 140K/mm2) and also The PF and Q30 reads are about 70%. I wonder what i coudl do to improve both cluster density and reads quality. If you have any tips please shout!
Comment
-
We've had poor assembly with nextera genomes above 62%, but decent assembly with the same genomes prepped with truseq.
I've never run high gc on nextseq, just have been told that we need to spike in at least 20% phiX (compared to 5% recommended for high gc on miseq/hiseq)Microbial ecologist, running a sequencing core. I have lots of strong opinions on how to survey communities, pretty sure some are even correct.
Comment
Latest Articles
Collapse
-
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 -
-
by seqadmin
The field of conservation genomics centers on applying genomics technologies in support of conservation efforts and the preservation of biodiversity. This article features interviews with two researchers who showcase their innovative work and highlight the current state and future of conservation genomics.
Avian Conservation
Matthew DeSaix, a recent doctoral graduate from Kristen Ruegg’s lab at The University of Colorado, shared that most of his research...-
Channel: Articles
03-08-2024, 10:41 AM -
ad_right_rmr
Collapse
News
Collapse
Topics | Statistics | Last Post | ||
---|---|---|---|---|
Started by seqadmin, Yesterday, 06:37 PM
|
0 responses
12 views
0 likes
|
Last Post
by seqadmin
Yesterday, 06:37 PM
|
||
Started by seqadmin, Yesterday, 06:07 PM
|
0 responses
10 views
0 likes
|
Last Post
by seqadmin
Yesterday, 06:07 PM
|
||
Started by seqadmin, 03-22-2024, 10:03 AM
|
0 responses
51 views
0 likes
|
Last Post
by seqadmin
03-22-2024, 10:03 AM
|
||
Started by seqadmin, 03-21-2024, 07:32 AM
|
0 responses
68 views
0 likes
|
Last Post
by seqadmin
03-21-2024, 07:32 AM
|
Comment