Can I pool sequencing libraries with different insert sizes on the AVITI™ system?

Question

Can I pool sequencing libraries with different insert sizes on the AVITI or AVITI24™ system?

Answer

Yes, but consider using Individually Addressable Lanes (IAL) or adjusting pooling to account for differences in efficiency between library sizes and types. Equimolar pooling of libraries works best when the libraries have similar size distributions. Due to differences in efficiency, when libraries of disparate size distributions are pooled together (equimolar), the resulting sequencing read distribution can be uneven. Smaller library sizes tend to be over-represented, and larger libraries tend to be under-represented.

Summary

• Equimolar pooling of libraries with similar size distributions (within ~200 bp of each other) generally results in a similar read distribution in the final output
• Libraries of different fragment size distributions can vary in their efficiency during sequencing, so equimolar pooling can result in unequal read distribution in the final output
• You can adjust pooling for libraries with different size distributions to compensate for efficiency differences and achieve the desired sequencing output for each library

Considerations

Size Bias and Pooling Strategy

If a library contains a broad fragment size distribution, you can perform a dual-sided bead cleanup to narrow the fragment size range and reduce size bias. Follow the instructions provided by the bead manufacturer for this process.

Figure 1. (A) Linear Elevate RNA-seq libraries prepared in five different size distributions within 200 bp of each other. (B) Pooled equimolar and sequenced with Cloudbreak™ and Cloudbreak Freestyle™.

For libraries with similar, overlapping size distributions (within ~200 bp), equimolar pooling generally results in a similar read distribution in the final sequencing output (Figure 1). See Which sequencing recipe should I use for my library insert size? to ensure you select the appropriate recipe for optimal results.

However, libraries of different fragment sizes can vary in their efficiency during sequencing when they are pooled together. Due to their size, smaller libraries make more copies during rolling circle amplification than larger libraries and are thus brighter and easier to detect. As a result of this size bias, libraries of different sizes pooled at equimolar concentrations might not be equally represented in the final sequencing output (Figure 2).

For pools made from libraries with different size distributions, adjusting pooling ratios helps compensate for bias and achieve the desired read distribution (Figure 2). In general, the larger the discrepancy (and less overlap) in library sizes that are pooled together, the more pronounced the effect on the final read distribution. As a result, pooling libraries with more disparate size ranges requires greater adjustment to achieve equal read distributions in the final output.

Figure 2. (A) Linear PCR-plus and Pippin size-selected libraries. (B) Equimolar and adjusted pooling with Cloudbreak Freestyle.

Individually Addressable Lanes

If libraries have compatible sequencing requirements (e.g., run configuration), you can load them using Individually Addressable Lanes (IAL) to avoid size bias introduced during pooling. See the AVITI System User Guide for specifics on IAL compatibility requirements, and use the Cloudbreak Sequencing User Guide to adjust input for the library size that you run in each lane. You can also sequence pools using IAL. However, pool and run only like-sized libraries in each lane unless you adjust pooling ratios.

PhiX Representation

PhiX representation also changes slightly depending on the size of the libraries it is pooled with. When used as a spike-in with much smaller libraries, PhiX tends to have reduced representation in the final sequencing output. When used as a spike-in with much larger libraries, PhiX tends to have increased representation in the final sequencing output. See the Cloudbreak Sequencing User Guide for PhiX spike-in recommendations.