Editing BioMicroCenter:Oxford Nanopore Technologies (section)

==Oxford Nanopore Sequencing==
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  !Service 
  !Nanopore Sequencing
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  |INPUT || Nanopore libraries
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  |MIN CONCENTRATION || Dependent on library type, read count not guaranteed
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  |INCLUDED SERVICES 
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* Quality Control:UV-vis measurement
* Nanopore Sequencing 
* Demultiplexing
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  |ADDITIONAL SERVICES ||
* Quality Control
* [[BioMicroCenter:NanoPore_Library_Prep|Nanopore library preparation]]
* Modified basecalling available on request
* Reference FASTA genome and BED regions file required for adaptive sampling
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  |DATA FORMATS 
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*FASTQ (stored 90 d and archived) 
*FAST5 (stored 30 d and deleted) 
*Assorted Nanopore QC documents
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  |PRICING || [[BioMicroCenter:Pricing#LONG_READ_LIBRARIES|NANOPORE LIBRARIES]] and [[BioMicroCenter:Pricing#LONG_READ_SEQUENCING|NANOPORE SEQUENCING]] 
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  |SUBMISSION 
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* MIT - [https://mit.ilabsolutions.com/service_item/new/3381?spt_id=3863 ilabs] 
* External - [[BioMicroCenter:Forms|form]]
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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.  
<br><br>
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.
<br><br>
Contaminants known to cause issues for the nanopores even at low amounts include: <br>
<li>EDTA<br><li>ethanol<br><li>isopropanol<br><li>NaCl<br><li>guanidinium chloride<br><li>guanidinium isothiocyanate<br><li>phenol<br> 
<br><br>
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. 
<br><br>
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.
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[[Image:Nanopore.jpg|300px|middle|Ji, CM et al (2024). Viruses, 16(5), 798.]] <br><br>
[[Image:Spanreads.jpg|200px|middle|Page Lab assembled ultra long read gDNA data showing a long read spanning Illumina contigs]] <br><br>
[[Image:IGV_vimentin.jpg|300px|middle|IGV spanning the Vimentin gene with assembled amplified cDNA]]
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