Asoberan: Create consolidated long read sequencing page (ONT PromethION, PacBio Revio)

2026-04-21T01:49:33Z

Create consolidated long read sequencing page (ONT PromethION, PacBio Revio)

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{{BioMicroCenter}}

The MIT BioMicro Center offers long-read sequencing on two platforms: Oxford Nanopore Technologies PromethION P2 Solo and PacBio Revio. Both platforms excel at applications where read length matters — de novo assembly, structural variant detection, full-length isoform sequencing, and epigenetic base modification — but differ significantly in chemistry, accuracy profile, throughput, and library requirements.

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'''Choosing a platform?''' Jump to [[#PLATFORM_COMPARISON|platform comparison]] below, or contact [mailto:biomicro@mit.edu biomicro@mit.edu].
</div>

== OVERVIEW ==

{| class="wikitable" style="width:100%; text-align:center;"
|-
! style="width:18%; background:#dce8f5;" | PLATFORM
! style="width:22%; background:#dce8f5;" | OUTPUT / FLOWCELL
! style="width:20%; background:#dce8f5;" | TYPICAL READ LENGTH
! style="width:20%; background:#dce8f5;" | AVAILABILITY
|-
| [[Image:PromethION.jpg|center|120px]] '''PromethION P2 Solo'''
| ~50–150 Gb (up to 290 Gb theoretical max)
| Variable; N50 typically 10–50 kb Ultra-long reads >100 kb possible
| In-house; MIT and external users
|-
| [[Image:Sequel.jpeg|center|120px]] '''PacBio Revio'''
| ~80 Gb HiFi per SMRTcell
| HiFi: typically 8-16kb kb 
| style="font-size:90%; color:#555;" | Via collaboration with nearby academic cores.
|}

== SEQUENCING PLATFORMS ==



== Oxford Nanopore Technologies ==


The BioMicro Center operates two [https://nanoporetech.com/products/sequence/promethion-2-solo PromethION P2 Solo] instruments from [https://nanoporetech.com Oxford Nanopore Technologies], each capable of running two flowcells concurrently. Nanopore sequencing passes DNA or RNA through a protein pore under voltage; current fluctuations as bases traverse the pore are decoded in real time into sequence and base modification calls. Unlike optical platforms, there is no theoretical read-length limit — the BMC has seen reads exceeding 300 kbp. The platform natively detects base modifications (methylation, hydroxymethylation, and others) without bisulfite conversion or additional chemistry. BMC runs each flowcell is run to completion and not reused.

=== PromethION P2 Solo — Sequencing ===

{|
|- style="vertical-align:top;"
| style="width:370px;" |
{| class="wikitable" border=1
! Service
! Nanopore Sequencing
|-
| INPUT || Completed Nanopore libraries
|-
| MIN CONCENTRATION || Library-type dependent; read count not guaranteed
|-
| INCLUDED SERVICES
|
* Quality Control: UV-vis measurement
* Nanopore Sequencing
* Demultiplexing
* Modified basecalling available on request
* Adaptive sampling (FASTA + optional BED file required)
|-
| ADDITIONAL SERVICES ||
* [[BioMicroCenter:NanoPore_Library_Prep|Nanopore library preparation]]
|-
| DATA FORMATS
|
* FASTQ (stored 90 days, then archived)
* FAST5 / POD5 (stored 30 days, then deleted)
* Assorted Nanopore QC
|-
| PRICING || [[MIT:Pricing]] · [[BioMicroCenter:Pricing]]
|-
| SUBMISSION
|
* MIT – [https://mit.ilabsolutions.com/service_item/new/3381?spt_id=3863 iLabs]
* External – [[BioMicroCenter:Forms|form]]
|-
|}
|

'''Typical output:''' ~50 Gb for standard genomic DNA (sheared and size-selected); up to ~150 Gb for metagenomic or short-fragment libraries; theoretical maximum ~290 Gb per flowcell. Output is highly variable and depends on library quality and input DNA integrity. Read count is not guaranteed. 

'''Run duration:''' Up to 72 hours per flowcell (adjustable). Adaptive sampling and real-time basecalling occur during the run. 

'''Modified basecalling:''' Native DNA or RNA sequencing captures base modification information (5mC, 5hmC, 6mA, and others) without additional sample preparation. Not all modifications can be called in a single run with current chemistry; trace data (FAST5/POD5) can be re-basecalled with different models. Re-basecalling can be arranged as a separate bioinformatics project. 

'''Adaptive sampling:''' Enriches or depletes specific genomic regions in real time without physical selection steps. Users must supply at least a FASTA reference genome and optionally a BED file of target regions. Target regions should represent 0.1–10% of expected reads for enrichment or depletion to be effective. 

'''Contaminants:''' The following are known to damage or inhibit nanopores even at low concentrations and must be removed prior to submission:
* EDTA
* Alcohols (ethanol, isopropanol)
* High concentrations of salts (NaCl, guanidinium chloride, guanidinium isothiocyanate)
* Phenol
|}

=== PromethION Library Preparation ===

The BioMicro Center supports three standard library preparation kits for Nanopore sequencing.

{| class="wikitable" style="width:100%; text-align:left;"
|-
! style="background:#dce8f5; width:15%;" | Kit
! style="background:#dce8f5; width:20%;" | Method
! style="background:#dce8f5; width:65%;" | Details
|-
| '''LSK114''' (SQK-LSK114)
| Ligation-based dsDNA
|
* '''Input:''' 1 µg clean dsDNA, >10 µL (50-100fmol)
* '''QC:''' UV-vis measurement; Final QC by Qubit
* '''Best for:''' Standard genomic DNA, flexible fragment sizes
* End repair → A-tail → adapter ligation → SPRI size selection
* Barcoding extension (SQK-NBD114) available for multiplexing up to 96 samples
|-
| '''RBK114''' (SQK-RBK114)
| Tagmentation-based dsDNA
|
* '''Input:''' 400 ng clean dsDNA, >30 kbp preferred, >10 µL
* '''QC:''' UV-vis and FemtoPulse; Final QC by Qubit
* '''Best for:''' Ultra-long read applications; largest fragment sizes
* Tagmentation with adapters
* For ultra-long reads: microgram input, no size selection; reads >300 kbp observed but rare; overall yield is lower.
|-
| '''RNA004''' (SQK-RNA004)
| Direct RNA sequencing
|
* '''Input:''' 1ug total RNA or 350 ng clean polyA+ mRNA (not total RNA), >10 µL
* '''QC:''' UV-vis and Fragment Analyzer; Final QC by Qubit
* '''Best for:''' Full-length transcript sequencing, isoform detection, RNA base modifications
* First-strand cDNA synthesis (optional but improves yield) → adapter ligation
|}



== PacBio ==


PacBio Revio sequencing is available through BMC's collaboration with local academic core facilities. The Revio uses [https://www.pacb.com/technology/hifi-sequencing/ HiFi (CCS) sequencing]: a single SMRTbell library molecule is sequenced multiple times end-to-end in a rolling fashion, and the resulting subreads are assembled into a high-fidelity consensus read with very high accuracy. This error correction is built into the sequencing run itself — unlike Nanopore, which achieves high accuracy through consensus polishing after sequencing. Library preparation is performed at the BMC; sequencing is performed at a partner core.

=== Revio — Sequencing ===

{|
|- style="vertical-align:top;"
| style="width:370px;" |
{| class="wikitable" border=1
! Service
! Revio Sequencing
|-
| INPUT || Completed SMRTbell libraries
|-
| MIN VOLUME || 12 µL
|-
| MIN CONCENTRATION || >1 nM ''(1 nM ≈ 0.65 ng/µL/kb insert)''
|-
| INCLUDED SERVICES
|
* Sample QC on [[BioMicroCenter:QC#AATI_FEMTO_PULSE|FemtoPulse]]
* Sequencing
* Primary analysis (CCS/HiFi)
* Data storage for 1 year
|-
| ADDITIONAL SERVICES ||
* [[BioMicroCenter:PacBio_Library_Preparation|PacBio library preparation]]
* [[BioMicroCenter:PippinPrep|Pippin Prep]] for size selection
|-
| PRICING || [[MIT:Pricing]] · [[BioMicroCenter:Pricing]]
|-
| SUBMISSION
|
* MIT – [https://mit.ilabsolutions.com/service_item/new/3381?spt_id=9912 iLabs]
* External – [[BioMicroCenter:Forms|form]]
|-
| PROVIDED THROUGH || Local academic core collaboration
|-
|}
|

'''How it works:''' The Revio loads ~10 M SMRTbell molecules into an array of zero-mode waveguides (ZMWs) by Poissonian distribution. Fluorescently labeled nucleotides are incorporated by a single polymerase tethered in each ZMW; incorporation events are detected in real time. Because the SMRTbell is circular, the polymerase reads the same molecule multiple times, generating subreads that are collapsed into a HiFi read with >>Q20 accuracy. Typical HiFi read lengths are 10–20 kb. 

'''Output:''' ~80 Gb HiFi per SMRTcell. 

'''Base modifications:''' Because the Revio measures polymerase kinetics (dwell time per base), it can detect base modifications that alter incorporation kinetics — including bacterial methylation (6mA, 4mC) and mammalian 5mC in CpG context. CpG methylation calling is included in standard HiFi analysis.
|}

=== Revio — Library Preparation ===

PacBio library preparation is performed at the BioMicro Center using the SMRTbell Prep Kit v3.0. Pricing includes quality control prior to prep.

{| class="wikitable" style="width:100%; text-align:left;"
|-
! style="background:#dce8f5; width:15%;" | Application
! style="background:#dce8f5; width:20%;" | Method
! style="background:#dce8f5; width:65%;" | Details
|-
| '''Genomic DNA'''
| SMRTbell Prep Kit v3.0
|
* '''Input:''' 150 ng–5 µg clean dsDNA, unsheared preferred at 50–60 kbp, >10 µL
* '''QC:''' UV-vis and FemtoPulse; Final QC by Qubit
* Single-strand overhang removal → damage repair → end repair → A-tailing → SMRTbell A/T ligation → two rounds of SPRI clean
* If average fragment size >50–60 kbp, Covaris gTube shearing available to target ~8-12 kbp
* Size selection verified by Fragment Analyzer or FemtoPulse
|}

== PLATFORM COMPARISON ==


{| class="wikitable" style="width:100%; text-align:center;"
|-
! style="background:#dce8f5;" | SPEC
! style="background:#e6f4ea;" | ONT PromethION P2
! style="background:#e8f0fe;" | PacBio Revio
|-
| '''Sequencing principle'''
| Ionic current through protein nanopore
| Real-time fluorescent SBS (ZMW array)
|-
| '''Read accuracy'''
| Raw reads: ~Q20; consensus polished: >Q30 (99.9%)
| HiFi reads: ~Q40 on-instrument; no post-run polishing required
|-
| '''Typical read length'''
| N50: 10–50 kb for standard gDNA; ultra-long reads >100 kb achievable
| HiFi: 10-20 kb typical
|-
| '''Output / flowcell'''
| 50–150 Gb typical (290 Gb theoretical max)
| ~80 Gb HiFi per SMRTcell
|-
| '''Run duration'''
| Up to 72 hours (adjustable)
| ~24 hours
|-
| '''Native base modification'''
| Yes — broad (5mC, 5hmC, 6mA, and more) no additional prep required
| Yes — kinetics-based (6mA, 4mC, 5mC CpG) included in standard HiFi analysis
|-
| '''Direct RNA sequencing'''
| Yes (SQK-RNA004)
| No
|-
| '''Adaptive sampling'''
| Yes — real-time enrichment/depletion
| No
|-
| '''Multiplexing'''
| Up to 96 barcodes/flowcell (NBD114)
| Limited — indexing practical mainly for amplicons
|-
| '''Instrument link'''
| [https://nanoporetech.com/products/sequence/promethion-2-solo PromethION P2 Solo]
| [https://www.pacb.com/revio/ PacBio Revio]
|}

=== Strengths and best use cases ===

'''Choose ONT PromethION when:'''
* Ultra-long reads are needed (>50 kb, e.g. spanning repeats or structural variants)
* Native base modification profiling beyond CpG (6mA, 5hmC, etc.)
* Direct RNA sequencing without cDNA conversion
* Adaptive sampling / real-time target enrichment or depletion
* High multiplexing (up to 96 samples/flowcell)
* Rapid turnaround is needed (real-time data during run)

'''Choose PacBio Revio when:'''
* High single-read accuracy is required without post-run polishing
* Full-length isoform sequencing (IsoSeq) with highly accurate reads
* De novo assembly of moderate-complexity genomes with accurate long reads
* CpG methylation calling is needed alongside variant calling

== FAQ ==

=== Can I get my base modification data? ===
Yes for both platforms. ONT detects a broad range of modifications natively; modified basecalling is available on request and can be applied to archived FAST5/POD5 data. PacBio CpG methylation calling is included in standard HiFi primary analysis.

=== What is adaptive sampling and do I need to do anything special? ===
Adaptive sampling is an ONT-only feature that uses the sequencer to eject molecules in real time based on a reference sequence, enriching or depleting specific genomic regions without physical library manipulation. To use it, provide at least a FASTA reference genome (and optionally a BED file of target regions) at submission. Target regions should represent 0.1–10% of expected reads.

=== What contaminants should I avoid for ONT? ===
EDTA, alcohols (ethanol, isopropanol), high-salt buffers (NaCl, guanidinium chloride, guanidinium isothiocyanate), and phenol will damage nanopores even at low concentrations and must be removed before submitting libraries. Contact [mailto:biomicro@mit.edu biomicro@mit.edu] if you are unsure about your elution buffer.

== SUBMISSION ==

All sequencing and library prep is submitted through [https://mit.ilabsolutions.com/service_item/new/3381?spt_id=3863 iLabs] (MIT users) or via the [[BioMicroCenter:Forms|external submission form]]. Contact [mailto:biomicro@mit.edu biomicro@mit.edu] before submitting non-standard samples or to discuss project design.

BioMicroCenter:LongRead - Revision history

Asoberan: Create consolidated long read sequencing page (ONT PromethION, PacBio Revio)