BioMicroCenter:Oxford Nanopore Technologies

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The MIT BioMicro Center has two PromethION P2 Solos. 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 each unit can theoretically run up to 2 flowcells simultaneously depending on the projects.

Oxford Nanopore Sequencing[edit]

Service	Nanopore Sequencing
INPUT	Nanopore libraries
MIN CONCENTRATION	Dependent on library type, read count not guaranteed
INCLUDED SERVICES	Quality Control:UV-vis measurement Nanopore Sequencing Demultiplexing
ADDITIONAL SERVICES	Quality Control Nanopore library preparation Modified basecalling available on request Reference FASTA genome and BED regions file required for adaptive sampling
DATA FORMATS	FASTQ (stored 90 d and archived) FAST5 (stored 30 d and deleted) Assorted Nanopore QC documents
PRICING	NANOPORE LIBRARIES and NANOPORE SEQUENCING
SUBMISSION	MIT - ilabs External - form

A variety of preparation kits allow DNA 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, amplifying should be simple, thus a lower concentration submission is reasonable.

Nanopore library contains adaptor with both motor protein and tether. If tether interacts with a nanopore successfully, the motor protein will shift the molecule into and through the protein nanopore. The voltage set across the nanopore shifts 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 and/or fastq.

Contaminants known to cause issues for the nanopores even at low amounts include:

EDTA

ethanol

isopropanol

NaCl

guanidinium chloride

guanidinium isothiocyanate

phenol

Modified basecalling is available on request. Not all desired basecalled modifications are possible in a single sequencing run with the current chemistries and basecalling software, but trace data (fast5) can be taken and re-basecalled using different basecallers. Re-basecalling can be done as a separate bioinformatic project.

For adaptive sampling, the user must provide at least a FASTA genome file and, optionally a BED file with the region(s) of interest. It is important that for both enrichment and depletion such region(s) only entail at maximum 10% of the genome.

Oxford Nanopore Sequencer[edit]

SPEC	PromethiON P2 Solo
SEQUENCER	© Oxford Nanopore Technologies
AVG OUTPUT/FLOWCELL	50 Gb
MAX TIME/FLOWCELL	3 days (adjustable)
BEST FOR	LONGEST READS COUNTING APPLICATIONS