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This may be a silly question, but I've been struggling to find the answer. I'll be using the Nextera dual indexing kit to prepare a single sample for genome sequencing. We're intending to sequence this sample without pooling it with anything else. The Illumina protocol for a 1-plex design like I've described says that I can use any single I7 adapter and any single i5 adapter. When I'm multiplexing samples, I need to worry about base diversity in my indices. Why is this not a concern when I'm not multiplexing??
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There are two issues for diversity...for the index reads (when multiplexing), and the diversity of the standard reads over the entire population of molecules being sequenced.
If you are multiplexing, you need to ensure the component indicies in the run satisfy several characteristics (having a large edit distane between them, being in both detection cameras, etc), to ensure robust detection of clusters and unambiguous assignment of reads to samples.
Without multiplexing, you don't care about the index read because you're not going to do one. However the generic diversity requirement is still important format of the same reasons; if the library was all one amplicon, for example, every cluster would be illuminated at the exact same base in every cycle and the cluster detection software will be very unhappy (and likely unable to focus and/or detect clusters). People typically spike in some amount of phiX library or other diverse sample.
Good luck. -
I was confused because I thought that at least one index was read as part of Read 1 or Read 2. From looking over the FAQ on the Illumina website, I can see that this is not the case and like you said, we have the option of not reading the indices. Thanks!Originally posted by ECO View PostComment
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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...06-18-2026, 07:11 AM -
Data variability is still an issue in sequencing technologies despite the advances in reproducibility and accuracy of these platforms. But the problem does not originate in the sequencing itself, but in the previous steps, before the sample reaches the sequencer.
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.
...06-02-2026, 10:05 AM
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