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Old 07-12-2016, 01:01 AM   #1
StephanK
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Location: Europe

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Default SmartSeq2 on Illumina HiSeq 4000

Hi!

I am new to the field and I try to establish single cell transcriptome sequencing with the Fluidigm C1 machine. We already decided that we use the SmartSeq2 protocol from Clontech (SmartSeq v4). Now our Institute got rid of several Illumina HiSeq 2000 machines that are recommended to use with the Nextera XT librabry prep that is recommended from Clontech. We do have several HiSeq 4000 machines and after talking with the facility they mentioned I should fractionate the cDNA on a Kovaris (with ultrasound) and then use the NEBNext ChIP Seq Mastermix for Illumina sequencing. Do you think this is the best protocol or is there another available? Of course I checked online but that did not yield much... I checked several papers for single cell sequencing and practically all of them use the Illumina 2000 sequencer. Is there a specific non-obvious reason for that? Is it only because of the Nextera XT Kit?

Unrelated to the SmartSeq2 protocol, can one use UMIs with the Kovaris ultrasound shredding of the cDNA? Will there not be many UMIs lost since they are always at the end of the molecule, so they are on smaller cDNA fragments. Are smaller fragments then more likely to be lost during purification processes?

Thank you very much in advance!

Stephan
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Old 07-12-2016, 12:44 PM   #2
pmiguel
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Location: Purdue University, West Lafayette, Indiana

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Hi Stephan,
Would you really sonicate nearly 96 cDNA samples? The whole idea behind using Nextera is that the transposable element fragments the DNA for you. That makes it possible to construct 96 or 384 libraries at a time.

The main problems with running C1 libraries on a HiSeq 4000 would be:

(1) Apparently the 3000/4000 doesn't do so well with with large insert libraries. They can jump the walls of the patterned flowcell?! Not really an issue, I would think as Nextera doesn't tend to make particularly large insert libraries.
Worst case, just do a size select on your library pool prior to clustering.

(2) Illumina sales reps told me that HiSeq 3000/4000 SBS kits come ready to do dual indexing. Nextera 96 and 384-library construction kits use dual indexing. But if the other lanes on the HiSeq flowcell use single indexed libraries, the sequencing core may not want to do the run as a dual indexed one. And the flowcells have 8 lanes.
Whereas the HiSeq 2500 Rapid flowcells only have 2 lanes. So running a dual indexed run on one of them is fine.
Still, I bet running single and dual indexed lanes on the HiSeq 3000/4000 would work. Just run them all as dual and demultiplex off-instrument twice. Once for the dual indexed lanes and once for the single indexed lanes.

Finally, I would recommend your contacting Fluidigm. I'm sure they have experience running their samples on a HiSeq3000/4000

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Phillip
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