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Old 01-08-2018, 07:28 AM   #1
kiruseq
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Default scRNAseq with Illumina SureCell kit - PE or single reads?

Hi everyone,
this may be a bit of a dumb question but I'm afraid I'm fairly new to the field.

So, for an upcoming project we're planning to use the Illumina SureCell kit for single cell RNAseq and I've just been looking into which sequencing platform might be appropriate for our needs. I've been trying to calculate how many cells I can sequence per flow cell to meet our desired sequencing depth of around 300,000 reads/cell. However, now I'm unsure whether I should base my calculation on the specified single or pair end reads per run, since with the SureCell library, read1 only covers the cell specific barcode and UMI and read2 covers the actual cDNA insert.

For example the NextSeq High Output kit suggests around 400M reads for single, and 800M reads for PE per run respectively. I'm inclined to "disregard" read 1 and base my calculation on single reads which would give me around 1,300 cells/run. Can anybody please tell me if this is the right approach? Or should I base my calculation on pair end reads?

Thanks a lot for the help!
/K
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Old 01-08-2018, 07:48 AM   #2
misterc
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Hi,

Yes, you should be basing your calculations by disregarding read 1. Illumina is essentially double-counting as you'll get 400M clusters per flow cell on a High Output kit (dependent on density).
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Old 01-08-2018, 07:58 AM   #3
kiruseq
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Thank you!
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