Sequencing

For BD^® SMK libraries, why do I need more reads for different types of cells rather than for the same type?

The Sample Tag targets have different expression levels among different cell types. Most of the reads will go to the predominant cell type if there are various cell types within a sample. We have validated that 600 reads/cell should be enough to recover most of the cells within a sample having various cell types. When you are working with samples with one cell type such as cell lines or samples for which the expression level of the Sample Tag target among the different cell types is similar, 120 reads/cell should be enough to recover most of the cells within the sample.

I want to sequence my BD Rhapsody™ Libraries on a NovaSeq™ Sequencer. Can I use the 100 cycle kit?

Yes, our pipeline can accommodate shorter read lengths for WTA or Targeted assays to take the advantage of the 100 cycle kit on the NovaSeq™ Sequencer. The acceptable criteria for valid R1 and R2 reads is 60 bases for R1, the entirety of which should match the cell barcode and the UMI, and 42 for R2, which contains the cDNA sequences.

Are BD Rhapsody™ Libraries compatible with BGI™ sequencing platforms?

Yes, theoretically, Illumina™ libraries can be converted to BGI™ libraries and sequenced on BGI™ sequencing platforms such as the MGISEQ-2000.

Can BD Rhapsody™ Libraries be sequenced with other types of libraries on Illumina platforms?

The read-length requirement for BD Rhapsody™ Libraries is PE75. Note the read-length requirement for other libraries to be run together. If mixing with other types of libraries is required, we recommend that the other libraries be of high diversity. If not, increase at least the recommended amount of PhiX for the sequencer used.

Can single end sequencing be used for BD Rhapsody™ Libraries, for example, 150 x 1?

No, we only support paired-end sequencing with a minimum 75 bp on both sides.

How much sequencing is sufficient?

Sequencing depth is dependent on application. For cell type clustering, shallow sequencing is sufficient. For in-depth analysis, such as comparison across multiple libraries, deep sequencing is recommended. We recommend meeting the requirement for recursive substitution error correction (RSEC), sequencing depth of ≥6 to reach the threshold of sequencing saturation where most molecules of the library have been recovered. For detailed recommendations, refer to the Sequencing Depths Recommendation section in your specific protocol.