, printed on 04/19/2024
For sequencing Targeted Spatial Gene Expression libraries, use the same sequencing parameters recommended for their parent Visium Spatial Gene Expression libraries.
The recommended sequencing depth for Spatial Targeted Gene Expression libraries based on pre-designed panels is 5,000 to 10,000 sequenced read pairs per tissue-covered spot.
Sequencing recommendations can be optimized for a particular gene panel and sample type when a whole transcriptome analysis is available from either the unenriched (untargeted) Spatial Gene Expression library or from a similar sample. The whole transcriptome data can be analyzed using the targeted-depth tool in Space Ranger by supplying the appropriate “target panel CSV” file and the results of a spaceranger count run. The spaceranger targeted-depth tool provides sequencing depth recommendations based on the fraction of reads in the whole transcriptome library that map to genes included in the panel.
At sequencing depths higher than the recommended levels, some samples may exhibit a reduced signal-to-noise ratio in UMI counts due to the presence of low-abundance PCR artifacts. For this reason, the spaceranger count pipeline will subsample Spatial Targeted Gene Expression libraries that have been sequenced to a depth exceeding 15,000 reads under tissue per spot. Only reads associated with barcodes of tissue-covered spots are counted towards this sequencing depth limit.
While changing this limit is not recommended, the maximum depth after subsampling may be adjusted or disabled using the --rps-limit argument to spaceranger count.