10x Genomics Support/Space Ranger 2.1/Analysis/

Sequencing Recommendations for Visium

10x recommends sequencing read 2 to a length of 90 bases.

Space Ranger v1.1 and later is less sensitive to read length effects. The rate at which reads map confidently to a gene, as well as the sensitivity of the resulting analysis, is consistent across a range of read lengths longer than 70 bp. This graph shows the relationship between read mapping and read 2 length, after trimming, for six representative samples.

Mapped reads are consistent across read lengths over 70 bp after trimming:

10x Genomics recommends sequencing read 2 to a length of 50 bases.

For formalin fixed paraffin embedded (FFPE) tissue the construct is of a known length. Since the reads are aligned to the reference, read 2 length shorter than 50 bases can lead to decreased performance.