PromethION: Protocol

Ligation sequencing gDNA - automated Hamilton NGS STAR 96 with Multiplex Ligation Sequencing Kit XL (SQK-MLK111.96-XL) V MLKH_9145_v111_revM_15Dec2021

Barcoding of native genomic DNA libraries

Requires the Multiplex Ligation Sequencing Kit XL
Automation of library preparation
Increase reproducibility
No PCR required
Features 96 unique barcodes
Enables low-plex sequencing
Allows analysis of native DNA
High sequencing output

For Research Use Only

This is a Legacy product This kit is soon to be discontinued and we recommend all customers to upgrade to the latest chemistry for their relevant kit which is available on the Store. If customers require further support for any ongoing critical experiments using a Legacy product, please contact Customer Support via email: support@nanoporetech.com.

FOR RESEARCH USE ONLY

概要

Barcoding of native genomic DNA libraries

Requires the Multiplex Ligation Sequencing Kit XL
Automation of library preparation
Increase reproducibility
No PCR required
Features 96 unique barcodes
Enables low-plex sequencing
Allows analysis of native DNA
High sequencing output

For Research Use Only

Document version: MLKH_9145_v111_revM_15Dec2021

1. Overview of the protocol

重要

This is a Legacy product

This kit is soon to be discontinued and we recommend all customers to upgrade to the latest chemistry for their relevant kit which is available on the Store. If customers require further support for any ongoing critical experiments using a Legacy product, please contact Customer Support via email: support@nanoporetech.com. For further information on please see the product update page.

Automated Multiplex Ligation Sequencing Kit XL features:

This kit is recommended for users who:

Would like to process multiple samples simultaneously, either with a multichannel pipette or a liquid handling robot
Want to optimise their sequencing experiment for output
Wish to low-plex samples for Whole Genome Sequencing (WGS)
Need a PCR-free method of multiplexing to preserve additional information, such as base modifications
Require control over read length
Would like to utilise upstream processes, such as size selection or whole genome amplification

重要

To use this automated method, method installation and training is required by Oxford Nanopore Technologies. For more information, please contact your local representative.

Introduction to the automated Multiplex Ligation Sequencing Kit XL protocol

The Multiplex Ligation Sequencing Kit XL (SQK-MLK111.96-XL) is designed to enable low-plex sequencing. Oxford Nanopore Technologies has written an internal script which enables the liquid handling robot to carry out native barcoding of genomic DNA using this sequencing kit. We currently allow the multiplexing of two samples on a flow cell or three samples on two flow cells.

To efficiently load multiple PromethION Flow Cells, we recommend using the Loading multiple PromethION Flow Cells protocol as a guideline.

Steps in the sequencing workflow: Prepare for your experiment You will need to:

Extract your DNA, and check its length, quantity and purity. The quality checks performed during the protocol are essential in ensuring experimental success.
Ensure you have your sequencing kit, the correct equipment and third-party reagents
Download the software for acquiring and analysing your data
Check your flow cell to ensure it has enough pores for a good sequencing run

Automated library preparation

You will need to:

Off-deck:

Prepare your sample input plate off-deck

On-deck:

Repair the DNA, and prepare the DNA ends for adapter attachment
Ligate Native barcodes supplied in the kit to the DNA ends
Ligate sequencing adapters supplied in the kit to the DNA ends

Off-deck:

Prime the flow cell, and load your DNA library into the flow cell

Sequencing

You will need to:

Start a sequencing run using the MinKNOW software, which will collect raw data from the device and convert it into basecalled reads
Demultiplex barcoded reads in MinKNOW or the Guppy basecalling
Start the EPI2ME software and select a workflow for further analysis (this step is optional)

重要

We do not recommend mixing barcoded libraries with non-barcoded libraries prior to sequencing.

Timings

96 samples with 2 samples per flow cell

Process	Minutes per step
End Repair and Adenylation	56
Native Barcode Ligation	75
Adapter Ligation	95
Total	226

96 samples with 3 samples across 2 flow cells

Process	Minutes per step
End Repair and Adenylation	56
Native Barcode Ligation	73
Adapter Ligation	78
Total	207

重要

Optional fragmentation and size selection

By default, the protocol contains no DNA fragmentation step, however in some cases it may be advantageous to fragment your sample. For example, when working with lower amounts of input gDNA (100 ng – 500 ng), fragmentation will increase the number of DNA molecules and therefore increase throughput. Instructions are available in the DNA Fragmentation section of Extraction methods.

Additionally, we offer several options for size-selecting your DNA sample to enrich for long fragments - instructions are available in the Size Selection section of Extraction methods.

重要

Compatibility of this protocol

This protocol should only be used in combination with:

Multiplex Ligation Sequencing Kit XL (SQK-MLK111.96-XL)
R9.4.1 flow cells (FLO-PRO002)
Flow Cell Wash Kit (EXP-WSH004)

2. Equipment and consumables

材料

Multiplex Ligation Sequencing Kit XL (SQK-MLK111.96-XL)
1200 ng gDNA per sample

消耗品

NEB Blunt/TA Ligase Master Mix (NEB, M0367)
NEBNext® Quick Ligation Reaction Buffer (NEB, B6058)
NEBNext FFPE Repair Mix (NEB, M6630)
NEBNext Ultra II End repair/dA-tailing Module (NEB, E7546)
NEBNext Quick Ligation Module (NEB, E6056)
Nuclease-free water (e.g. ThermoFisher, AM9937)
nuclease-free waterで調整した 80% エタノール溶液
Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
1.5 ml Eppendorf DNA LoBind tubes
2.0 ml Eppendorf DNA LoBind tubes
Agencourt AMPure XP beads (Beckman Coulter™ cat # A63881)
Qubit™ Assay Tubes (Invitrogen, Q32856)
Hamilton 50 µl CO-RE tips with filter (Cat# 235948)
Hamilton 300 µl CO-RE tips with filter (Cat# 235903)
Hamilton 1000 µl CO-RE tips with filter (Cat# 235905)
Hamilton PCR ComfortLid (Cat# 814300)
Bio-Rad Hard-Shell® 96-Well PCR Plates (Cat# HSP9601)
Hamilton 60 ml Reagent Reservoir, Self-Standing with Lid (Cat# 56694-01)

装置

P1000 pipette and tips
P200 pipette and tips
P20 pipette and tips
アイスバケツ（氷入り）
タイマー
Qubit fluorometer (or equivalent)
Hamilton NGS STAR 96 (NGS STAR with Multi-Probe Head 96)
Hamilton On-Deck Thermal Cycler (ODTC)
Hamilton MTP landscape carrier (cat# 182365)
Hamilton Ambion magnet adapter (cat# 10107866)

オプション装置

Agilent Bioanalyzer (or equivalent)
Eppendorf 5424 centrifuge (or equivalent)

For this protocol, you will need 1200 ng gDNA per sample for R9.4.1 flow cells.

We recommend using 80 ng/µl per sample with a minimum of 15 µl per well when preparing the input plate.

インプットDNA

インプットDNAのQC方法

インプットDNAの量と品質の要件を満たすことが重要です。DNAの使用量が少なすぎたり多すぎたり、あるいは品質の低いDNA（例としてDNAが非常に断片化されていたり、RNAや化学汚染物質が含まれている場合など）を使用すると、ライブラリーの調製に影響を及ぼす可能性があります。

DNAサンプルの品質管理の方法については、Input DNA/RNA QC protocolのプロトコルをご覧ください。

コンタミネーション

DNAの抽出する方法によっては、精製DNAに特定の化学汚染物質が残留する可能性があり、ライブラリ調製の効率やシークエンシングの品質に影響を及ぼす可能性があります。コンタミネーションについての詳細は、コミュニティーの Contaminants page をご覧ください。

Input workfile

Input workfiles are required prior to running this protocol on the Hamilton NGS STAR 96.

Oxford Nanopore Technologies will provide a template of the input workfiles in an Excel file.

In the DATA tab of the file, there will be the template for the library preparation data
In the INFO tab, there will be the template for the identification data

Library preparation data example:

Identification data example:

Output workfiles will be generated. Below are examples of the output workfiles after a run through of the method.

Library preparation data example:

Identification data example:

Hamilton NGS STAR 96 and deck layout

This method has been tested and validated using the Hamilton NGS STAR 96 (with Multi-Probe Head 96), Hamilton Ambion magnet adapter, Hamilton MTP landscape carrier and Hamilton On-Deck Thermal Cycler (ODTC).

Deck layout

The deck layout has been updated from the standard layout. Please see the "Prepare the deck" step of this protocol for more details.

Data tracking

For data tracking purposes, we have included the option to add user ID before starting any process. This can be filled using any method the user prefers.

We recommend using barcode stickers to track the input and output plates for data tracking. These can be tracked on the workfile and entered on the UI alongside the reagent lot barcodes when prompted.

Convenient reagent kits are available on request from NEB for the Multiplex Ligation Sequencing Kit XL.

This will contain the appropriate NEB reagents and the required volumes for the protocol on the Hamilton NGS STAR 96. For more information from NEB, please see "Find Products for Nanopore Sequencing".

Multiplex Ligation Sequencing Kit XL (SQK-MLK111.96-XL) contents

Name	Acronym	Cap colour	Number of vials	Fill volume per vial (µl)
Adapter Mix II T	AMII T	Green	1	320
Sequencing Buffer II	SBII	Red	4	1,500
Loading Beads II	LBII	Pink	4	1,500
Loading Solution	LS	White cap, pink sticker	4	1,500
EDTA	EDTA	Clear	1	700
Elution Buffer	EB	15 ml bottle	1	10,000
Long Fragment Buffer	LFB	30 ml bottle	1	20,000
Flush Buffer XL	FB	30 ml bottle	6	15,500
Flush Tether	FLT	White cap, purple sticker	2	1,600
Native Barcodes	NB01-96	N/a	1 plate	8 µl per well

Consumables and equipment quantities:

Consumables	No. of consumables for all conditions
Hamilton 50 µl CO-RE tips with filter	960
Hamilton 300 µl CO-RE tips with filter	960
Hamilton 1000 µ CO-RE tips with filter	96
Bio-Rad Hard-Shell® 96-well PCR Plate	8

This protocol requires the tip decks to be completely filled before starting a run. Partially filled tip decks will cause an error with the liquid handling robot.

We recommend using Hamilton tips for efficient liquid handling.

Reagent quantities:

Note: Volumes for x48 samples will be available soon.

Full method

Reagents	x96 samples
80% ethanol	80 ml
AMPure XP Beads	15 ml
Nuclease-free water	10 ml
Long Fragment Buffer (LFB)	2 bottles
Elution Buffer (EB)	1 bottle
EDTA	1 vial
Native Barcode plate	1 plate
Adapter Mix F (AMII F)	1 vial
NEBNext FFRE DNA Repair Buffer	130 µl
NEBNext FFPE DNA Repair Mix	90 µl
Ultra II End Prep Reaction Buffer	130 µl
Ultra II End Prep Enzyme Mix	120 µl
Blunt/TA Ligase Master Mix	1210 µl
NEBNext Quick Ligation Reaction Buffer (5x)	590 µl
Quick T4 DNA Ligase	310 µl

__Multiple steps combined:__ ### End Repair and Adenylation step to Native Barcode Ligation step

Reagents	x96 samples
80% ethanol	80 ml
AMPure XP Beads	15 ml
Nuclease-free water	10 ml
NEBNext FFRE DNA Repair Buffer	130 µl
NEBNext FFPE DNA Repair Mix	90 µl
Ultra II End Prep Reaction Buffer	130 µl
Ultra II End Prep Enzyme Mix	120 µl
Blunt/TA Ligase Master Mix	1210 µl
EDTA	1 vial
Native Barcode plate	1 plate

### Native Barcode Ligation step to Adapter Ligation step

Reagents	x96 samples
80% ethanol	40 ml
AMPure XP Beads	15 ml
Nuclease-free water	10 ml
Long Fragment Buffer (LFB)	2 bottles
Elution Buffer (EB)	1 bottle
Blunt/TA Ligase Master Mix	1210 µl
EDTA	1 vial
Native Barcode plate	1 plate
Adapter Mix F (AMII F)	1 vial
NEBNext Quick Ligation Reaction Buffer (5x)	590 µl
Quick T4 DNA Ligase	310 µl

__Individual steps:__ #### End Repair step

Reagents	x96 samples
80% ethanol	40 ml
AMPure XP Beads	15 ml
Nuclease-free water	10 ml
NEBNext FFRE DNA Repair Buffer	130 µl
NEBNext FFPE DNA Repair Mix	90 µl
Ultra II End Prep Reaction Buffer	130 µl
Ultra II End Prep Enzyme Mix	120 µl

### Native Barcode Ligation step

Reagents	x96 samples
80% ethanol	40 ml
AMPure XP Beads	15 ml
Nuclease-free water	10 ml
Blunt/TA Ligase Master Mix	1210 µl
EDTA	1 vial
Native Barcode plate	1 plate

#### Adapter Ligation step

Reagents	x96 samples
AMPure XP Beads	15 ml
Long Fragment Buffer (LFB)	2 bottles
Elution Buffer (EB)	1 bottle
NEBNext Quick Ligation Reaction Buffer (5x)	590 µl
Quick T4 DNA Ligase	310 µl

Native barcode sequences

Component	Forward sequence	Reverse sequence
NB01	CACAAAGACACCGACAACTTTCTT	AAGAAAGTTGTCGGTGTCTTTGTG
NB02	ACAGACGACTACAAACGGAATCGA	TCGATTCCGTTTGTAGTCGTCTGT
NB03	CCTGGTAACTGGGACACAAGACTC	GAGTCTTGTGTCCCAGTTACCAGG
NB04	TAGGGAAACACGATAGAATCCGAA	TTCGGATTCTATCGTGTTTCCCTA
NB05	AAGGTTACACAAACCCTGGACAAG	CTTGTCCAGGGTTTGTGTAACCTT
NB06	GACTACTTTCTGCCTTTGCGAGAA	TTCTCGCAAAGGCAGAAAGTAGTC
NB07	AAGGATTCATTCCCACGGTAACAC	GTGTTACCGTGGGAATGAATCCTT
NB08	ACGTAACTTGGTTTGTTCCCTGAA	TTCAGGGAACAAACCAAGTTACGT
NB09	AACCAAGACTCGCTGTGCCTAGTT	AACTAGGCACAGCGAGTCTTGGTT
NB10	GAGAGGACAAAGGTTTCAACGCTT	AAGCGTTGAAACCTTTGTCCTCTC
NB11	TCCATTCCCTCCGATAGATGAAAC	GTTTCATCTATCGGAGGGAATGGA
NB12	TCCGATTCTGCTTCTTTCTACCTG	CAGGTAGAAAGAAGCAGAATCGGA
NB13	AGAACGACTTCCATACTCGTGTGA	TCACACGAGTATGGAAGTCGTTCT
NB14	AACGAGTCTCTTGGGACCCATAGA	TCTATGGGTCCCAAGAGACTCGTT
NB15	AGGTCTACCTCGCTAACACCACTG	CAGTGGTGTTAGCGAGGTAGACCT
NB16	CGTCAACTGACAGTGGTTCGTACT	AGTACGAACCACTGTCAGTTGACG
NB17	ACCCTCCAGGAAAGTACCTCTGAT	ATCAGAGGTACTTTCCTGGAGGGT
NB18	CCAAACCCAACAACCTAGATAGGC	GCCTATCTAGGTTGTTGGGTTTGG
NB19	GTTCCTCGTGCAGTGTCAAGAGAT	ATCTCTTGACACTGCACGAGGAAC
NB20	TTGCGTCCTGTTACGAGAACTCAT	ATGAGTTCTCGTAACAGGACGCAA
NB21	GAGCCTCTCATTGTCCGTTCTCTA	TAGAGAACGGACAATGAGAGGCTC
NB22	ACCACTGCCATGTATCAAAGTACG	CGTACTTTGATACATGGCAGTGGT
NB23	CTTACTACCCAGTGAACCTCCTCG	CGAGGAGGTTCACTGGGTAGTAAG
NB24	GCATAGTTCTGCATGATGGGTTAG	CTAACCCATCATGCAGAACTATGC
NB25	GTAAGTTGGGTATGCAACGCAATG	CATTGCGTTGCATACCCAACTTAC
NB26	CATACAGCGACTACGCATTCTCAT	ATGAGAATGCGTAGTCGCTGTATG
NB27	CGACGGTTAGATTCACCTCTTACA	TGTAAGAGGTGAATCTAACCGTCG
NB28	TGAAACCTAAGAAGGCACCGTATC	GATACGGTGCCTTCTTAGGTTTCA
NB29	CTAGACACCTTGGGTTGACAGACC	GGTCTGTCAACCCAAGGTGTCTAG
NB30	TCAGTGAGGATCTACTTCGACCCA	TGGGTCGAAGTAGATCCTCACTGA
NB31	TGCGTACAGCAATCAGTTACATTG	CAATGTAACTGATTGCTGTACGCA
NB32	CCAGTAGAAGTCCGACAACGTCAT	ATGACGTTGTCGGACTTCTACTGG
NB33	CAGACTTGGTACGGTTGGGTAACT	AGTTACCCAACCGTACCAAGTCTG
NB34	GGACGAAGAACTCAAGTCAAAGGC	GCCTTTGACTTGAGTTCTTCGTCC
NB35	CTACTTACGAAGCTGAGGGACTGC	GCAGTCCCTCAGCTTCGTAAGTAG
NB36	ATGTCCCAGTTAGAGGAGGAAACA	TGTTTCCTCCTCTAACTGGGACAT
NB37	GCTTGCGATTGATGCTTAGTATCA	TGATACTAAGCATCAATCGCAAGC
NB38	ACCACAGGAGGACGATACAGAGAA	TTCTCTGTATCGTCCTCCTGTGGT
NB39	CCACAGTGTCAACTAGAGCCTCTC	GAGAGGCTCTAGTTGACACTGTGG
NB40	TAGTTTGGATGACCAAGGATAGCC	GGCTATCCTTGGTCATCCAAACTA
NB41	GGAGTTCGTCCAGAGAAGTACACG	CGTGTACTTCTCTGGACGAACTCC
NB42	CTACGTGTAAGGCATACCTGCCAG	CTGGCAGGTATGCCTTACACGTAG
NB43	CTTTCGTTGTTGACTCGACGGTAG	CTACCGTCGAGTCAACAACGAAAG
NB44	AGTAGAAAGGGTTCCTTCCCACTC	GAGTGGGAAGGAACCCTTTCTACT
NB45	GATCCAACAGAGATGCCTTCAGTG	CACTGAAGGCATCTCTGTTGGATC
NB46	GCTGTGTTCCACTTCATTCTCCTG	CAGGAGAATGAAGTGGAACACAGC
NB47	GTGCAACTTTCCCACAGGTAGTTC	GAACTACCTGTGGGAAAGTTGCAC
NB48	CATCTGGAACGTGGTACACCTGTA	TACAGGTGTACCACGTTCCAGATG
NB49	ACTGGTGCAGCTTTGAACATCTAG	CTAGATGTTCAAAGCTGCACCAGT
NB50	ATGGACTTTGGTAACTTCCTGCGT	ACGCAGGAAGTTACCAAAGTCCAT
NB51	GTTGAATGAGCCTACTGGGTCCTC	GAGGACCCAGTAGGCTCATTCAAC
NB52	TGAGAGACAAGATTGTTCGTGGAC	GTCCACGAACAATCTTGTCTCTCA
NB53	AGATTCAGACCGTCTCATGCAAAG	CTTTGCATGAGACGGTCTGAATCT
NB54	CAAGAGCTTTGACTAAGGAGCATG	CATGCTCCTTAGTCAAAGCTCTTG
NB55	TGGAAGATGAGACCCTGATCTACG	CGTAGATCAGGGTCTCATCTTCCA
NB56	TCACTACTCAACAGGTGGCATGAA	TTCATGCCACCTGTTGAGTAGTGA
NB57	GCTAGGTCAATCTCCTTCGGAAGT	ACTTCCGAAGGAGATTGACCTAGC
NB58	CAGGTTACTCCTCCGTGAGTCTGA	TCAGACTCACGGAGGAGTAACCTG
NB59	TCAATCAAGAAGGGAAAGCAAGGT	ACCTTGCTTTCCCTTCTTGATTGA
NB60	CATGTTCAACCAAGGCTTCTATGG	CCATAGAAGCCTTGGTTGAACATG
NB61	AGAGGGTACTATGTGCCTCAGCAC	GTGCTGAGGCACATAGTACCCTCT
NB62	CACCCACACTTACTTCAGGACGTA	TACGTCCTGAAGTAAGTGTGGGTG
NB63	TTCTGAAGTTCCTGGGTCTTGAAC	GTTCAAGACCCAGGAACTTCAGAA
NB64	GACAGACACCGTTCATCGACTTTC	GAAAGTCGATGAACGGTGTCTGTC
NB65	TTCTCAGTCTTCCTCCAGACAAGG	CCTTGTCTGGAGGAAGACTGAGAA
NB66	CCGATCCTTGTGGCTTCTAACTTC	GAAGTTAGAAGCCACAAGGATCGG
NB67	GTTTGTCATACTCGTGTGCTCACC	GGTGAGCACACGAGTATGACAAAC
NB68	GAATCTAAGCAAACACGAAGGTGG	CCACCTTCGTGTTTGCTTAGATTC
NB69	TACAGTCCGAGCCTCATGTGATCT	AGATCACATGAGGCTCGGACTGTA
NB70	ACCGAGATCCTACGAATGGAGTGT	ACACTCCATTCGTAGGATCTCGGT
NB71	CCTGGGAGCATCAGGTAGTAACAG	CTGTTACTACCTGATGCTCCCAGG
NB72	TAGCTGACTGTCTTCCATACCGAC	GTCGGTATGGAAGACAGTCAGCTA
NB73	AAGAAACAGGATGACAGAACCCTC	GAGGGTTCTGTCATCCTGTTTCTT
NB74	TACAAGCATCCCAACACTTCCACT	AGTGGAAGTGTTGGGATGCTTGTA
NB75	GACCATTGTGATGAACCCTGTTGT	ACAACAGGGTTCATCACAATGGTC
NB76	ATGCTTGTTACATCAACCCTGGAC	GTCCAGGGTTGATGTAACAAGCAT
NB77	CGACCTGTTTCTCAGGGATACAAC	GTTGTATCCCTGAGAAACAGGTCG
NB78	AACAACCGAACCTTTGAATCAGAA	TTCTGATTCAAAGGTTCGGTTGTT
NB79	TCTCGGAGATAGTTCTCACTGCTG	CAGCAGTGAGAACTATCTCCGAGA
NB80	CGGATGAACATAGGATAGCGATTC	GAATCGCTATCCTATGTTCATCCG
NB81	CCTCATCTTGTGAAGTTGTTTCGG	CCGAAACAACTTCACAAGATGAGG
NB82	ACGGTATGTCGAGTTCCAGGACTA	TAGTCCTGGAACTCGACATACCGT
NB83	TGGCTTGATCTAGGTAAGGTCGAA	TTCGACCTTACCTAGATCAAGCCA
NB84	GTAGTGGACCTAGAACCTGTGCCA	TGGCACAGGTTCTAGGTCCACTAC
NB85	AACGGAGGAGTTAGTTGGATGATC	GATCATCCAACTAACTCCTCCGTT
NB86	AGGTGATCCCAACAAGCGTAAGTA	TACTTACGCTTGTTGGGATCACCT
NB87	TACATGCTCCTGTTGTTAGGGAGG	CCTCCCTAACAACAGGAGCATGTA
NB88	TCTTCTACTACCGATCCGAAGCAG	CTGCTTCGGATCGGTAGTAGAAGA
NB89	ACAGCATCAATGTTTGGCTAGTTG	CAACTAGCCAAACATTGATGCTGT
NB90	GATGTAGAGGGTACGGTTTGAGGC	GCCTCAAACCGTACCCTCTACATC
NB91	GGCTCCATAGGAACTCACGCTACT	AGTAGCGTGAGTTCCTATGGAGCC
NB92	TTGTGAGTGGAAAGATACAGGACC	GGTCCTGTATCTTTCCACTCACAA
NB93	AGTTTCCATCACTTCAGACTTGGG	CCCAAGTCTGAAGTGATGGAAACT
NB94	GATTGTCCTCAAACTGCCACCTAC	GTAGGTGGCAGTTTGAGGACAATC
NB95	CCTGTCTGGAAGAAGAATGGACTT	AAGTCCATTCTTCTTCCAGACAGG
NB96	CTGAACGGTCATAGAGTCCACCAT	ATGGTGGACTCTATGACCGTTCAG

3. Computer requirements and software

PromethION 24/48 IT requirements

The PromethION device contains all the hardware required to control up to 24 (for the P24 model) or 48 (for the P48 model) sequencing experiments and acquire the data. The device is further enhanced with high performance GPU technology for real-time basecalling. Read more in the PromethION IT requirements document.

PromethION 2 Solo IT requirements

The PromethION 2 (P2) Solo is a device which directly connects into a GridION Mk1 or a stand-alone computer that meets the miminum specifications for real-time data streaming and analysis. Up to two PromethION flow cells can be can be run and each is independently addressable, meaning experiments can be run concurrently or individually. For information on the computer IT requirements, please see the PromethION 2 Solo IT requirements document.

Software for nanopore sequencing

MinKNOW

The MinKNOW software controls the nanopore sequencing device, collects sequencing data and basecalls in real time. You will be using MinKNOW for every sequencing experiment to sequence, basecall and demultiplex if your samples were barcoded.

For instructions on how to run the MinKNOW software, please refer to the MinKNOW protocol.

EPI2ME (optional)

The EPI2ME cloud-based platform performs further analysis of basecalled data, for example alignment to the Lambda genome, barcoding, or taxonomic classification. You will use the EPI2ME platform only if you would like further analysis of your data post-basecalling.

For instructions on how to create an EPI2ME account and install the EPI2ME Desktop Agent, please refer to this link.

フローセルのチェックをしてください

シークエンシング実験を開始する前に、フローセルのポアの数を確認することを強くお勧めします。このフローセルの確認は、MinION/GridION/PromethIONの場合は代理店への到着から１２週間以内に行ってください。またはFlongle Flow Cellの場合は代理店への到着から4週間以内に行う必要があります。Oxford Nanopore Technologiesは、フローセルチェックの実施から2日以内に結果が報告され、推奨される保管方法に従っていた場合に、以下の表に記載されているナノポアの有効数に満たさない場合には、フローセルを交換します。フローセルのチェックを行うには、Flow Cell Check documentの指示に従ってください。

Flow cell	保証する最小有効ポア数（以下の数未満のフローセルが交換対象となります）
Flongle Flow Cell	50
MinION/GridION Flow Cell	800
PromethION Flow Cell	5000

4. Prepare the deck

消耗品

1.5 ml Eppendorf DNA LoBind tubes
Hamilton 50 µl CO-RE tips with filter (Cat# 235948)
Hamilton 300 µl CO-RE tips with filter (Cat# 235903)
Hamilton 1000 µl CO-RE tips with filter (Cat# 235905)
Hamilton PCR ComfortLid (Cat# 814300)
Hamilton 60 ml Reagent Reservoir, Self-Standing with Lid (Cat# 56694-01)
Bio-Rad Hard-Shell® 96-Well PCR Plates (Cat# HSP9601)

装置

Hamilton NGS STAR 96 (NGS STAR with Multi-Probe Head 96)
Hamilton Ambion magnet adapter (cat# 10107866)
Hamilton MTP landscape carrier (cat# 182365)
Hamilton On-Deck Thermal Cycler (ODTC)

重要

Extra equipment required for the deck layout

Hamilton MTP landscape carrier (cat # 182365)
Hamilton Ambion magnet adapter (cat # 10107866)

Deck layout change

The deck layout has been updated from the standard NGS STAR 96 layout to improve the efficiency of the robot completing the protocol.

The changes will take approximately 3 minutes.

Final deck layout:

Plate stacker
Two tube racks

Positions 1 and 2 are indicated by the arrow below:

最終ステップ

Once the deck is correctly set up, the robot can be prepared to run the automation protocol.

5. Pre-processes

材料

1200 ng gDNA per sample
Long Fragment Buffer (LFB)

消耗品

nuclease-free waterで調整した 80% エタノール溶液
Nuclease-free water (e.g. ThermoFisher, cat #AM9937)
Hamilton 1000 µl CO-RE tips with filter (Cat# 235905)
Hamilton 300 µl CO-RE tips with filter (Cat# 235903)
Hamilton 50 µl CO-RE tips with filter (Cat# 235948)
Bio-Rad Hard-Shell® 96-Well PCR Plates (Cat# HSP9601)

Users have the option to use pre-process to complete automated upstream methods to prepare their samples.

最終ステップ

Once the process is complete, you will be returned to the method selection page, enabling the user to either start the library preparation process or another pre-process.