BioMicroCenter:Oxford Nanopore Technologies - Revision history

Slevine: /* Oxford Nanopore Sequencer */

2025-07-18T12:29:29Z

Oxford Nanopore Sequencer

← Older revision		Revision as of 12:29, 18 July 2025
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	\|-		\|-
	\| '''AVG OUTPUT/FLOWCELL'''<BR>		\| '''AVG OUTPUT/FLOWCELL'''<BR>
	\| 50 Gb		\| 50 Gb (highly variable)
	\|-		\|-
	\|'''MAX TIME/FLOWCELL'''		\|'''MAX TIME/FLOWCELL'''
	\| 3 days (adjustable)		\| 3 days (adjustable)
	\|}		\|}

Slevine at 11:52, 18 July 2025

2025-07-18T11:52:16Z

← Older revision		Revision as of 11:52, 18 July 2025
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	{{BioMicroCenter}}		{{BioMicroCenter}}
	<br><br>		<br><br>
	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~~.		The MIT BioMicro Center has two PromethION P2 Solos, each capable of running two concurrent flowcells. The Center supports all current standard ONT library prep protocols including both ligation and tagmentation methods as well as direct RNA measurement. Run protocols such as adaptive sampling are available. Each flowcell is run to completion and not reused - prices are for full flowcells.
	<br><br>		<br><br>
	==Oxford Nanopore Sequencing==		==Oxford Nanopore Sequencing==
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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~~.		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.
	<br><br>		<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.		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>		<br><br>
	Contaminants known to cause issues for the nanopores even at low amounts include: ~~<br>~~		Contaminants known to cause issues for the nanopores even at low amounts include:
	~~<li>~~EDTA~~<br><li>ethanol<br><li>isopropanol<br><li>~~NaCl~~<br><li>~~guanidinium chloride~~<br><li>~~guanidinium isothiocyanate~~<br><li>~~phenol~~<br>~~		*EDTA
			*Alcohols (EtOH, iPrOH
			*High concentrations of salts (NaCl, guanidinium chloride,guanidinium isothiocyanate
			*phenol
	<br><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.		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>		<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.~~		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 0.1-10% of expected reads
	\|		\|
	[[Image:Nanopore.jpg\|300px\|middle\|Ji, CM et al (2024). Viruses, 16(5), 798.]] <br><br>		[[Image:Nanopore.jpg\|300px\|middle\|Ji, CM et al (2024). Viruses, 16(5), 798.]] <br><br>
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	\|-		\|-
	\| '''AVG OUTPUT/FLOWCELL'''<BR>		\| '''AVG OUTPUT/FLOWCELL'''<BR>
	\|		\| 50 Gb
	50 Gb
	\|-		\|-
	\|'''MAX TIME/FLOWCELL'''		\|'''MAX TIME/FLOWCELL'''
	\|		\| 3 days (adjustable)
	3 days (adjustable)
	\|-
	~~\|'''BEST FOR'''~~
	\|
	*LONGEST READS
	*COUNTING APPLICATIONS
	\|}		\|}

Hilo1: /* Oxford Nanopore Sequencing */

2025-06-09T17:47:39Z

Oxford Nanopore Sequencing

← Older revision		Revision as of 17:47, 9 June 2025
Line 53:		Line 53:
	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.		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.
	\|		\|
	[[Image:Nanopore.jpg\|300px\|middle\|Ji, CM et al (2024). Viruses, 16(5), 798.]]		[[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]]		[[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]]		[[Image:IGV_vimentin.jpg\|300px\|middle\|IGV spanning the Vimentin gene with assembled amplified cDNA]]
	\|}		\|}

Hilo1: /* Oxford Nanopore Sequencing */

2025-06-09T17:47:00Z

Oxford Nanopore Sequencing

← Older revision		Revision as of 17:47, 9 June 2025
Line 54:		Line 54:
	\|		\|
	[[Image:Nanopore.jpg\|300px\|middle\|Ji, CM et al (2024). Viruses, 16(5), 798.]]		[[Image:Nanopore.jpg\|300px\|middle\|Ji, CM et al (2024). Viruses, 16(5), 798.]]
	[[Image:Spanreads.jpg\|~~150px~~\|middle\|Page Lab assembled ultra long read gDNA data showing a long read spanning Illumina contigs]]		[[Image:Spanreads.jpg\|200px\|middle\|Page Lab assembled ultra long read gDNA data showing a long read spanning Illumina contigs]]
	[[Image:IGV_vimentin.jpg\|300px\|middle\|IGV spanning the Vimentin gene with assembled amplified cDNA]]		[[Image:IGV_vimentin.jpg\|300px\|middle\|IGV spanning the Vimentin gene with assembled amplified cDNA]]
	\|}		\|}

Hilo1: /* Oxford Nanopore Sequencing */

2025-06-09T17:46:43Z

Oxford Nanopore Sequencing

← Older revision		Revision as of 17:46, 9 June 2025
Line 54:		Line 54:
	\|		\|
	[[Image:Nanopore.jpg\|300px\|middle\|Ji, CM et al (2024). Viruses, 16(5), 798.]]		[[Image:Nanopore.jpg\|300px\|middle\|Ji, CM et al (2024). Viruses, 16(5), 798.]]
	[[Image:Spanreads.jpg\|~~300px~~\|middle\|Page Lab assembled ultra long read gDNA data showing a long read spanning Illumina contigs]]		[[Image:Spanreads.jpg\|150px\|middle\|Page Lab assembled ultra long read gDNA data showing a long read spanning Illumina contigs]]
	[[Image:IGV_vimentin.jpg\|300px\|middle\|IGV spanning the Vimentin gene with assembled amplified cDNA]]		[[Image:IGV_vimentin.jpg\|300px\|middle\|IGV spanning the Vimentin gene with assembled amplified cDNA]]
	\|}		\|}

Hilo1: /* Oxford Nanopore Sequencing */

2025-06-09T17:45:56Z

Oxford Nanopore Sequencing

← Older revision		Revision as of 17:45, 9 June 2025
Line 54:		Line 54:
	\|		\|
	[[Image:Nanopore.jpg\|300px\|middle\|Ji, CM et al (2024). Viruses, 16(5), 798.]]		[[Image:Nanopore.jpg\|300px\|middle\|Ji, CM et al (2024). Viruses, 16(5), 798.]]
			[[Image:Spanreads.jpg\|300px\|middle\|Page Lab assembled ultra long read gDNA data showing a long read spanning Illumina contigs]]
			[[Image:IGV_vimentin.jpg\|300px\|middle\|IGV spanning the Vimentin gene with assembled amplified cDNA]]
	\|}		\|}

Hilo1: /* Oxford Nanopore Sequencer */

2025-06-09T17:39:25Z

Oxford Nanopore Sequencer

← Older revision		Revision as of 17:39, 9 June 2025
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	\|[[Image:PromethION.jpg\|center\|200px]]© Oxford Nanopore Technologies		\|[[Image:PromethION.jpg\|center\|200px]]© Oxford Nanopore Technologies
	\|-		\|-
	\| '''AVG ~~READS~~/FLOWCELL'''<BR>		\| '''AVG OUTPUT/FLOWCELL'''<BR>
	\|		\|
	50 Gb		50 Gb

Hilo1: /* Oxford Nanopore Sequencing */

2025-06-09T17:33:06Z

Oxford Nanopore Sequencing

← Older revision		Revision as of 17:33, 9 June 2025
Line 53:		Line 53:
	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.		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.
	\|		\|
	[[Image:Nanopore.jpg\|300px\|middle]]		[[Image:Nanopore.jpg\|300px\|middle\|Ji, CM et al (2024). Viruses, 16(5), 798.]]
	\|}		\|}

Hilo1: /* Oxford Nanopore Sequencing */

2025-06-09T17:28:29Z

Oxford Nanopore Sequencing

← Older revision		Revision as of 17:28, 9 June 2025
Line 51:		Line 51:
	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.		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>		<br><br>
	For adaptive sampling, the user must provide ~~both~~ a ~~FASTQ~~ genome file and 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.		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.
	\|		\|
	[[Image:Nanopore.jpg\|300px\|middle]]		[[Image:Nanopore.jpg\|300px\|middle]]

Hilo1: /* Oxford Nanopore Sequencing */

2025-05-20T19:22:05Z

Oxford Nanopore Sequencing

← Older revision		Revision as of 19:22, 20 May 2025
Line 42:		Line 42:
	\|}		\|}
	\|		\|
	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.		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>		<br><br>
	~~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.		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>		<br><br>
	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~~.		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 both a FASTQ genome file and 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.
	\|		\|
	[[Image:Nanopore.jpg\|300px\|middle]]		[[Image:Nanopore.jpg\|300px\|middle]]