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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!
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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. -
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.Originally posted by thiNGS View PostComment
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What's the %GC of these organisms? you may need to spike in a lot of PhiX(20%) if it's highMicrobial ecologist, running a sequencing core. I have lots of strong opinions on how to survey communities, pretty sure some are even correct.Comment
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Nextera works well up to 75% GC and does not require spike in for increasing sequence diversity.Comment
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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
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My comment is regarding sequencing metrics. I have not followed up assembly stats.Comment
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Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.07-01-2026, 11:43 AM
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I’m not a sequencing expert. I’m a purification scientist who uses NGS to evaluate workflows my group develops. With this perspective, we think about the sample first and the NGS workflow second. The sequencer is an exceptionally honest reporter, but it can only report on what you give it, so whether you get clean, interpretable data from an NGS workflow is largely determined before you begin.
Here are nine questions we think about, in roughly the order they matter, before...
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