Question

I would like to sequence 250 bases on Illumina Novaseq S4 on read 2. Kits of illumina come in the following sizes: can setup the sequencer for asymmetric sequencing, so that from a standard paired-end 2*100bp kit reads 150 bases from R1, and only 50 of the R2 (or vice versa).On SP kit they now support 2 x 250 bp, but not on S4.On the largest S4 kit it would be theoretically possible to sequence 250 + 50. The read quality at 150 bases is excellent, so I expect at least 200 useful bases.

​ Did anyone try this, or could point me to relevant information?Can you even specify such strong asymmetry in the software?Very importantly: Are there any risk of losing the lane?Are there any other risks (other than quality issues beyond 150 bases, which i assume I can easily trim away)? ps.: Cross posted onseqanswersfor a possibly more appropriate audience.

Answer 1

Yes, it is theoretically possible to achieve asymmetric sequencing on Illumina Novaseq S4 for 250 bases, with 150 bases from read 1 (R1) and 100 bases from read 2 (R2). However, specifying such strong asymmetry in the software may have limitations, and there could be risks, including potential lane loss. It's advisable to consult Illumina's technical support or community forums for specific guidance on implementing this approach.

Step-by-step explanation:

Sequencing 250 bases on Illumina Novaseq S4 with asymmetric reads involves optimizing the standard 2 x 100bp kit. In theory, with the largest S4 kit, you could sequence 250 bases by allocating 150 bases to R1 and 100 bases to R2.

The challenge lies in software configuration, as Illumina's sequencing software may not easily accommodate such extreme asymmetry. Illumina's user forums or technical support could provide insights into whether this specific setup has been attempted and how to navigate potential software constraints.

However, introducing such asymmetry may pose risks, including the possibility of lane loss. Illumina platforms are optimized for balanced read lengths, and deviating significantly from this may impact data quality and sequencing efficiency. It's crucial to weigh the benefits against potential drawbacks and seek guidance from the sequencing community to mitigate risks.

Additionally, consider the impact on downstream analysis, as tools may have assumptions about balanced reads. Despite the theoretical potential for obtaining 250 bases, practical implementation and associated risks should be thoroughly evaluated before proceeding with this sequencing strategy.