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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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The first step is collection, followed by preservation and sample preparation for analysis. Most scientists overlook those steps, but not being careful might just be skewing the experiment’s results.
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