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Hello all. Can anyone offer insight into the pros and cons of placing index barcodes at the 5' versus the 3' end of the read?
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5' of the read allows you to run multiplexed samples without doing the index read. I liked to do this on the GAII to avoid using the Paired-End module for SR multiplexing. Con is that the bases need to be balanced or you can throw off the cluster calling algorithms. Use cycle 5+ to avoid this type of problem.
3' end of read must mean the standard Illumina indexing method using a second priming and read to hit the index. That method works fine, extra reagents are in the SBS kits to allow for 7 cycles for the index read. Illumina only has 12-indexes for DNA and RNA out so far, so that would be a con. On the HiSeq this indexing read is easy to set up, I avoided it on the GAII. -
Thanks for the info!Comment
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Bioo has 48 barcodes using the 3' method. The kit works well.Comment
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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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