Nextera XT for WGS of bacteria

thiNGS · 02-02-2018, 02:54 AM

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!

JoeKutch · 02-02-2018, 12:41 PM

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.

nucacidhunter · 02-02-2018, 09:05 PM

Quote:

Originally Posted by thiNGS

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!

These two issues can have common or separate causes. To diagnose the issue it will be helpful if you can post run summary page (Reads tables) and also %Base from data by cycle plot of analysis page of SAV.

thermophile · 02-07-2018, 10:11 AM

What's the %GC of these organisms? you may need to spike in a lot of PhiX(20%) if it's high

nucacidhunter · 02-08-2018, 02:49 PM

Nextera works well up to 75% GC and does not require spike in for increasing sequence diversity.

thermophile · 02-09-2018, 02:35 PM

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)

nucacidhunter · 02-09-2018, 05:06 PM

My comment is regarding sequencing metrics. I have not followed up assembly stats.

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02-02-2018, 02:54 AM	#1
thiNGS Member Location: Netherlands Join Date: Sep 2014 Posts: 24	Nextera XT for WGS of bacteria 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!

02-07-2018, 10:11 AM	#4
thermophile Senior Member Location: CT Join Date: Apr 2015 Posts: 243	What's the %GC of these organisms? you may need to spike in a lot of PhiX(20%) if it's high __________________ Microbial ecologist, running a sequencing core. I have lots of strong opinions on how to survey communities, pretty sure some are even correct.

02-09-2018, 02:35 PM	#6
thermophile Senior Member Location: CT Join Date: Apr 2015 Posts: 243	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.

02-02-2018, 12:41 PM	#2
JoeKutch Member Location: Philadelphia Join Date: Oct 2016 Posts: 17	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) = 2.27pM*would 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.

02-08-2018, 02:49 PM	#5
nucacidhunter Jafar Jabbari Location: Melbourne Join Date: Jan 2013 Posts: 1,238	Nextera works well up to 75% GC and does not require spike in for increasing sequence diversity.

02-09-2018, 05:06 PM	#7
nucacidhunter Jafar Jabbari Location: Melbourne Join Date: Jan 2013 Posts: 1,238	My comment is regarding sequencing metrics. I have not followed up assembly stats.