The MIT BioMicro Center has one PromethION P24. We support de novo sequencing and have tested some RNA-seq metrics on these platforms. Each flowcell can potentially accommodate many barcoded samples (depending on sample quality and desired coverage) and the unit can theoretically run up to 24 flowcells simultaneously depending on the projects.
Oxford Nanopore Sequencing
Service
Nanopore Sequencing
INPUT
Nanopore libraries
MIN CONCENTRATION
Dependent on library type, read count not guaranteed
A variety of preparation kits allow DNA, cDNA, or direct RNA to be sequenced. The input amount necessary for each type of sample varies according to the desired result. For the longest reads of genomic DNA, micrograms worth of clean starting material provide the highest quality of data, but not the highest amounts of reads. For amplicons including cDNA made from RNA, amplifying hundreds of nanograms should be simple, thus a lower input in the picograms should be reasonable.
During library preparation, adaptor consisting of motor protein and tether is ligated to the ends of RNA or DNA with tether furthest from the template. Adaptors can also be bought with barcodes for multiplexing. If tether interacts with a nanopore successfully, motor protein will shift the molecule into and through the protein nanopore. A voltage is set across the nanopore and the current will shift as base pairs move through the pore. Nanopores are arranged in an array such that multiple molecules can be sequenced simultaneously. Sequencing and demultiplexing all occur in real time providing fast5 or fastq.
Contaminants known to cause issues for the nanopores so far include: EDTA, ethanol, isopropanol, NaCl, guanidinium chloride, guanidinium isothiocyanate, and phenol. The nanopores on the flowcell can be directly affected by low amounts of contamination.
