Microfluidics & sequencing prices

Microfluidics

Concept of microfluidics

• Carve hair-thick channels into a plastic (PBMS = Polydimethylsiloxane).
• Your samples travel through a labyrinth of channels with chambers.
• In each chamber (or section of channel) you can perform a reaction.

Adavantages of microfluidics

• Small volume
  • less reagent
  • less dilution of small samples
• Small space required
• Small thermal mass / fast heat transfer
• Fast mixing by diffusion
• Precise flow control (laminar flow)
• Protected from contamination
• Automation is easy
• … but its complicated…

Fabrication of microfluidics chips

Microfluidics for single-cell mRNA seq

Watch video from Dolomite - click here

Sequencing prices

Source: Ziegenhain 2017

• Drop-seq (690$) is the most cost-effective method when sequencing 254 cells at adepth of 250,000 reads, and
  • 2.7$ per cell
• SCRB-seq (810$),
• Mars-Seq (820$), and
• Smart-seq2 (1,090$, with in-house-produced Tn5 transposase)

Cost comparison of key methods

10X Genomics: 3’mRNA-Seq

• A new (2016) commercial chip+kit to sequence 10K’s of cells using microfluidics.
• Around 1,000 to 10,000 cells are captured in just 10 minutes.
• Workflow similar to DropSeq: capture cells with beads, release RNA, oligo-dT primed cDNA to get a 3’ mRNA-seq library.

“Economy of scales” for preparation but not for sequencing

Prices @ Cambridge facility https://genomics.cruk.cam.ac.uk/services/Single-cell

Costs: £1500ish per sample:

• £1200 per sample for the single-cell 3’mRNA-Seq library prep.
  • for 1000 cells 1.2£
  • for 10000 cells 0.12£
• £200 for 1000 cells at 50,000 reads each (assuming multiplexing samples in a HiSeq 4000 lane)
  • for 1000 cells @ 250K: 1000£
  • for 10000 cells @ 250K: 10000£

Slashing prices

• Multiplexing on the sequencing lane: There are different illumina index primers
• There is always a lot of trouble shooting: choose a method that can do a 100 cells for the first couple of pilot rounds.
• Use of in-house enzymes instead of commercial kits
• Gene specific primers: instead of the polyA primer, you can make a set of 100 gene specific primers to subset to genes
• Pull-down of ready libraries: You can try to remove genes from the ready library by pulldown (biotin labeled DNA primer complementary to say, Hemoglobin)