Quick Thoughts On Illumina Insert Size Limits

We were chatting about insert size limits in Illumina sequencing on the Discord today. So I decided to try and do a quick patent search. Looking at some of the older bridge amplification patents from Pascal Mayer.

Insert size is probably limited by a number of factors. One concern on patterned flowcells is that templates might form bridges between wells resulting in duplicate reads. With super-resolution, and well spacing going below 300nm this could be a factor.

However this doesn’t mean it’s the only factor. The original Isothermal patents were not on patterned flowcells. But also show a fragment length dependance on amplification:

In the figure above T90 is 90bp, T605 is 605bp etc. The Y-axis is normalized for template length and therefore gives a fluorescence based estimate of the cluster size.

At very short template length efficiency drops (they cite the notion that short templates are rigid and can’t bend as easily).

But beyond that as template length increases efficiency drops. I didn’t see a justification for this in the patent. I assume longer fragments are more likely to form secondary structure etc.

Illumina run a fixed cluster generation protocol. As such it seems possible that they might be able to grow clusters from longer fragments under different conditions. However as longer fragments are likely to form duplicates anyway (on patterned flowcells), they likely don’t want to support this. But perhaps a hacked recipe might allow longer insert sizes? (at the risk of duplicates).

gdub42 also dug out this really interesting paper which shows fragment length dependance on read quality (particularly apparent in the second read):

Would be really fun to hack around with recipes and how growing clusters longer/for more cycles might effect this…