There are commercial sequencing kits/sequencers that allow for paired end sequencing in which the two reads obtained for each fragment are of different length?

For example 150bp for R1 and 50bp for R2?

Is it at least possible in principle? There are some published speculations about that possibility?

  • $\begingroup$ it's used for certain applications, e.g. 10X single cell where R1 is shorter, being only the bar code and UMI $\endgroup$ Commented Jul 24, 2019 at 12:59
  • $\begingroup$ If you're looking for technologies that do this explicitly to optimize for something, it's possible in principle. If you're looking for an explanation why some files you're looking at are different, it may be due to some pre-processing on the files, such as in the scRNA-seq case $\endgroup$ Commented Jul 24, 2019 at 18:19
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    $\begingroup$ Yes. For Illumina kits for example you include R1 and R2 length in the sample sheet. As long as you don't exceed the maximum number of cycles you will be fine. For a 150 cycle kit you can perfectly run 75-75, 125-25 and even 50-10. $\endgroup$
    – Mack123456
    Commented Jul 25, 2019 at 20:21

1 Answer 1


As indicated in the comments, yes, you can definitely tell standard Illumina sequencers to sequence mates in a pair to different lengths. This is quite common in single-cell RNA-seq, where R1 is often just a cell barcode and an UMI, so sequencing it to 150 bases would be a huge waste or reagent. One caveat to this is that you can't buy reagents sized for these custom length, just things like 150 base PE or other standards. So unless you end up saving and pooling reagents after such runs you end up paying the same for projects with one shorter read than if you'd just directly sequenced everything to the same length. Given that sequencer runs only take a day or two at the moment, this also doesn't result in that immense of a time savings.


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