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

Oxford Nanopore Technologies
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PromethION: Protocol

Ligation sequencing gDNA - Multiplex Ligation Sequencing Kit XL (SQK-MLK111.96-XL) V MLK_9144_v111_revI_01Dec2021

Barcoding of native genomic DNA libraries

  • Requires the Multiplex Ligation Sequencing Kit XL
  • No PCR required
  • Features 96 unique barcodes
  • Enables low-plex sequencing
  • Allows analysis of native DNA

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

概览

Barcoding of native genomic DNA libraries

  • Requires the Multiplex Ligation Sequencing Kit XL
  • No PCR required
  • Features 96 unique barcodes
  • Enables low-plex sequencing
  • Allows analysis of native DNA

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.

Document version: MLK_9144_v111_revI_01Dec2021

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.

Multiplex Ligation Sequencing Kit XL features

This kit is recommended for users who:

  • 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

Introduction to the manual Multiplex Ligation Sequencing Kit XL protocol

This protocol describes how to carry out native barcoding of genomic DNA using the Multiplex Ligation Sequencing Kit XL (SQK-MLK111.96-XL). This kit is designed to enable low-plex sequencing and this manual protocol outlines how to sequence two samples across one flow cell. This enables users to sequence 96 samples across 48 flow cells to provide an easier workflow for whole genome sequencing (WGS).

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

Prepare your library

You will need to:

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

Native barcoding workflow1

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 software
  • Start the EPI2ME software and select a workflow for further analysis (this step is optional)
重要

我们不建议在测序前混合含条码文库与无条码文库。

重要

可选步骤：DNA片段化以及片段大小筛选

本实验手册不包含DNA片段化步骤。但在某些情况下，将样品片段化可能有助于您的实验。例如，当起始gDNA量较少时（100ng-500ng），将DNA片段化能扩充分子数量，从而提高通量。请参考：牛津纳米孔社区“Extraction methods”（提取方法）板块的 DNA片段化部分。

我们也提供了一些用于富集DNA样品中长片段的方法，请参考：牛津纳米孔社区“Extraction methods”(提取方法) 板块的 片段大小筛选部分。

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)
  • 1000 ng gDNA per sample
耗材
  • NEB Blunt/TA 连接酶预混液（NEB，M0367）
  • NEBNext®快速连接反应缓冲液（NEB，B6058）
  • NEBNext FFPE修复混合液（NEB，M6630）
  • NEBNext Ultra II 末端修复/ dA尾添加模块（NEB，E7546）
  • NEBNext 快速连接模块（NEB，E6056）
  • 1.5 ml Eppendorf DNA LoBind 离心管
  • 0.2 ml thin-walled PCR tubes
  • Qubit™ 分析管（Invitrogen, Q32856）
  • 无核酸酶水（如ThermoFisher，AM9937）
  • Freshly prepared 70% ethanol in nuclease-free water
  • Agencourt AMPure XP beads (Beckman Coulter™ cat # A63881)
  • Qubit dsDNA HS Assay（双链DNA高灵敏度检测）试剂盒（Invitrogen, Q32851）
仪器
  • Hula混匀仪（低速旋转式混匀仪）
  • 迷你离心机
  • 磁力架
  • 涡旋混匀仪
  • 热循环仪
  • P1000 移液枪和枪头
  • P200 移液枪和枪头
  • P100 移液枪和枪头
  • P20 移液枪和枪头
  • P10 移液枪和枪头
  • P2移液枪和枪头
  • 盛有冰的冰桶
  • 计时器
  • Qubit荧光计 （或用于质控检测的等效仪器）
可选仪器
  • Agilent生物分析仪（或等效仪器）
  • Eppendorf 5424 离心机（或等效器材）

For this protocol, you will need 1000 ng gDNA per sample for R9.4.1 flow cells.

起始DNA

DNA质控

选择符合质量和浓度要求的起始DNA至关重要的。使用过少或过多的DNA，或者质量较差的DNA（如，高度碎片化、含有RNA或化学污染物的DNA）都会影响文库制备。

有关如何对DNA样品进行质控，请参考起始DNA/RNA质控实验指南 。

化学污染物

从原始样本中提取DNA的方法不同，可能会导致经纯化的DNA中所残留的化学污染物不同。这会影响文库的制备效率和测序质量。请在牛津纳米孔社区的 Contaminants（污染物）页面 了解更多信息。

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

sqk-mlk111.96-xl 1

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

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. 计算机要求及软件

PromethION 24/48 的IT配置要求

PromethION设备的硬件能够同时控制多达24个（适用于P24型号）或48个（适用于P48型号）测序实验，并采集数据。此外，设备借助高性能GPU技术，可以实时识别碱基。 请参阅 PromethION IT 配置要求文档，了解更多信息。

PromethION 2 Solo 的IT配置要求

作为一款小型台式测序设备，PromethION 2 Solo可独立或同时运行两张测序芯片。您只需将PromethION 2 Solo连接到GridION Mk1或符合最低技术规格要求的独立计算机，即可实现数据的实时采集和分析。欲了解更多信息，请参阅PromethION 2 Solo IT 配置要求文档。

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.

测序芯片质检

我们强烈建议您在开始测序实验前，对测序芯片的活性纳米孔数进行质检。质检需在您收到MinION /GridION /PremethION测序芯片12周之内进行，或者在您收到Flongle测序芯片四周内进行。Oxford Nanopore Technologies会对活性孔数量少于以下标准的芯片进行替换** ：

测序芯片 芯片上的活性孔数确保不少于
Flongle 测序芯片 50
MinION/GridION 测序芯片 800
PromethION 测序芯片 5000

** 请注意：自收到之日起，芯片须一直贮存于Oxford Nanopore Technologies推荐的条件下。且质检结果须在质检后的两天内递交给我们。请您按照 测序芯片质检文档中的说明进行芯片质检。

4. DNA repair and end-prep

材料
  • 1000 ng gDNA per sample
耗材
  • NEBNext FFPE DNA 修复混合液（NEB，M6630）
  • NEBNext® Ultra II 末端修复/ dA尾添加模块（NEB，E7546）
  • Agencourt AMPure XP beads (Beckman Coulter™ cat # A63881)
  • 无核酸酶水（如ThermoFisher，AM9937）
  • Freshly prepared 70% ethanol in nuclease-free water
  • 0.2 ml薄壁PCR管
  • 1.5 ml Eppendorf DNA LoBind 离心管
  • Qubit dsDNA HS Assay（双链DNA高灵敏度检测）试剂盒（Invitrogen, Q32851）
  • Qubit™ 分析管（Invitrogen, Q32856）
仪器
  • P1000 移液枪和枪头
  • P100 移液枪和枪头
  • P10 移液枪和枪头
  • 热循环仪
  • 盛有冰的冰桶
  • 迷你离心机
  • Hula mixer (gentle rotator mixer)
  • 磁力架
  • Qubit荧光计 （或用于质控检测的等效仪器）
重要

可选步骤：DNA片段化以及片段大小筛选

本实验手册不包含DNA片段化步骤。但在某些情况下，将样品片段化可能有助于您的实验。例如，当起始gDNA量较少时（100ng-500ng），将DNA片段化能扩充分子数量，从而提高通量。请参考：牛津纳米孔社区“Extraction methods”（提取方法）板块的 DNA片段化部分。

我们也提供了一些用于富集DNA样品中长片段的方法，请参考：牛津纳米孔社区“Extraction methods”(提取方法) 板块的 片段大小筛选部分。

根据生产厂家的说明准备NEBNext FFPE DNA 修复混合液和 NEBNext Ultra II 末端修复/ dA尾添加模块，并置于冰上。

为获得最优表现，NEB建议如下：

  1. 于冰上解冻所有试剂。

  2. 轻弹并/或翻转各管，确保各试剂充分混匀。
    注意： 请切勿涡旋振荡 FFPE DNA修复混合液或 Ultra II末端修复酶混合物。

  3. 同一日内首次打开一管试剂前，请务必先将该管试剂瞬时离心。

  4. Ultra II 末端修复缓冲液和 FFPE DNA 修复缓冲液内可能出现少量沉淀。待此两管液体回复至室温后，使用移液枪上下吹打数次，打散沉淀；然后涡旋振荡30秒，以确保沉淀充分溶解。
    注意： 请务必涡旋振荡混匀缓冲液。

  5. FFPE DNA 修复缓冲液可能轻微泛黄，不影响使用。

重要

勿涡旋振荡NEBNext FFPE DNA 修复混合液或NEBNext Ultra II 末端修复酶混合物。

In clean 0.2 ml thin-walled PCR tubes, aliquot 1000 ng per sample.

Make up each sample to 12 µl using nuclease-free water. Mix gently by pipetting and spin down.

Combine the following components per sample:

Between each addition, pipette mix 10 - 20 times.

Reagent Volume
NEBNext FFPE DNA Repair Buffer 0.875 µl
Ultra II End-prep reaction buffer 0.875 µl
Ultra II End-prep enzyme mix 0.75 µl
NEBNext FFPE DNA Repair Mix 0.50 µl
Total 3 µl
提示

We recommend making up a mastermix for the total number of samples and adding 3 µl to each individual sample.

Mix well by pipetting and spin down in a centrifuge.

使用热循环仪，在20℃下孵育5分钟，然后在65℃下孵育5分钟。

Transfer each sample to clean 1.5 ml Eppendorf DNA LoBind tube.

Resuspend the Agencourt AMPure XP beads by vortexing.

Add 15 µl of resuspended Agencourt AMPure XP beads to each end-prep reaction and mix by flicking the tube.

将离心管置于Hula混匀仪（低速旋转式混匀仪）上室温孵育5分钟。

Prepare 500 μl of fresh 70% ethanol in nuclease-free water.

将样品瞬时离心，并静置于磁力架上待磁珠和液相分离。保持离心管在磁力架上不动，用移液枪吸去清液。

Keep the tube on the magnet and wash the beads with 200 µl of freshly prepared 70% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.

Repeat the previous step.

Briefly spin down and place the tubes back on the magnet. Pipette off any residual ethanol. Allow to dry for 30 seconds, but do not dry the pellet to the point of cracking.

Remove the tubes from the magnetic rack and resuspend the pellet in 10 µl nuclease-free water. Spin down and incubate for 2 minutes at room temperature.

Pellet the beads on a magnet until the eluate is clear and colourless.

Remove and retain 10 µl of eluate for each sample into clean 1.5 ml Eppendorf DNA LoBind tubes, individually.

Note: If users are having difficulty retaining 10 µl without disturbing the beads, 8.5 µl can be retained instead, allowing 1 µl for quantification and 7.5 µl to be taken forward into the Native Barcode Ligation step.

CHECKPOINT

Quantify 1 µl of each eluted sample using a Qubit fluorometer.

步骤结束

取等摩尔质量的各样本，用于稍后的免扩增条形码连接步骤。如需要，您也可以此时将样品置于4℃储存过夜。

5. Native barcode ligation

材料
  • 免扩增条形码（NB01-NB96）
  • EDTA（EDTA）
耗材
  • 无核酸酶水（如ThermoFisher，AM9937）
  • 新制备的70%乙醇（用无核酸酶水配制）
  • NEB Blunt/TA 连接酶预混液（NEB，M0367）
  • Agencourt AMPure XP Beads (Beckman Coulter™, A63881)
  • 1.5 ml Eppendorf DNA LoBind离心管
  • Qubit dsDNA HS Assay（双链DNA高灵敏度检测）试剂盒（Invitrogen, Q32851）
  • Qubit™ 分析管（Invitrogen, Q32856）
仪器
  • 磁力架
  • 涡旋混匀仪
  • Hula混匀仪（低速旋转式混匀仪）
  • 迷你离心机
  • 热循环仪
  • 盛有冰的冰桶
  • P1000 移液枪和枪头
  • P100 移液枪和枪头
  • P10 移液枪和枪头
可选仪器
  • Qubit荧光计（或用于质控检测的等效仪器）

Prepare third party reagents in accordance with manufacturer's instructions, and place on ice:

Thaw the native barcodes at room temperature. Use one barcode per sample. Individually mix the barcodes by pipetting, spin down, and place them on ice.

Select two unique barcodes for each pair of samples to be run together.

In clean 1.5 ml Eppendorf DNA LoBind tubes, add the reagents in the following order per sample:

Reagent Volume
End-prepped DNA 7.5 µl
Native barcode 2.5 µl
Blunt/TA Ligase Master Mix 10 µl
Total 20 µl

轻轻吹打以充分混匀，并瞬时离心。

室温下孵育20分钟。

Add 2 µl of EDTA to each tube and mix thoroughly by pipetting and spin down briefly.

Pool the barcoded samples in a clean 1.5 ml Eppendorf DNA LoBind tube.

Resuspend the Agencourt AMPure XP beads by vortexing.

Add 16 µl of Agencourt AMPure XP beads to the pooled reaction, and mix by pipetting.

将离心管置于Hula混匀仪（低速旋转式混匀仪）上室温孵育10分钟。

Prepare 500 µl of fresh 70% ethanol in nuclease-free water.

Spin down the sample and pellet on a magnet for 5 minutes. Keep the tube on the magnetic rack until the eluate is clear and colourless, and pipette off the supernatant.

Keep the tube on the magentic rack and wash the beads with 200 µl of freshly prepared 70% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.

重复上述步骤。

将离心管瞬时离心后置于磁力架上。用移液枪吸走残留的乙醇。让磁珠在空气中干燥约30秒，但不要干至表面开裂。

将离心管从磁力架上移开。将磁珠重悬于35µl的无核酸酶水中，轻弹离心管混匀。

37℃下孵育10分钟。请每两分钟轻弹离心管10秒以搅动样本，促进DNA洗脱。

将离心管静置于磁力架上，直到磁珠和液相分离，且洗脱液澄清无色。

将此35µl洗脱液转移至一支新的1.5ml Eppendorf DNA LoBind管中。

CHECKPOINT

Quantify 1 µl of eluted sample using a Qubit fluorometer.

步骤结束

连有条形码的DNA样本将用于稍后的接头连接及纯化步骤。如需要，您也可以此时将样品置于4℃储存过夜。

6. Adapter ligation and clean-up

材料
  • 长片段缓冲液（LFB）
  • 洗脱缓冲液（EB）
  • Adapter Mix II T (AMII T)
耗材
  • NEBNext®快速连接模块（NEB，E6056）
  • NEBNext®快速连接反应缓冲液（NEB，B6058）
  • 1.5 ml Eppendorf DNA LoBind 离心管
  • Agencourt AMPure XP Beads (Beckman Coulter™, A63881)
  • Qubit dsDNA HS Assay（双链DNA高灵敏度检测）试剂盒（Invitrogen, Q32851）
  • Qubit™ 分析管（Invitrogen, Q32856）
仪器
  • 迷你离心机
  • 磁力架
  • 涡旋混匀仪
  • Hula混匀仪（低速旋转式混匀仪）
  • 热循环仪
  • P1000 移液枪和枪头
  • P200 移液枪和枪头
  • P100 移液枪和枪头
  • P20 移液枪和枪头
  • P10 移液枪和枪头
  • 盛有冰的冰桶
  • Qubit荧光计 （或用于质控检测的等效仪器）

Thaw the Elution Buffer (EB) and NEBNext Quick Ligation Reaction Buffer (5x) at room temperature, mix by vortexing, spin down and place on ice. Check the contents of each tube are clear of any precipitate.

Spin down the Quick T4 Ligase and the Adapter Mix II T (AMII T), and place on ice.

To enrich for DNA fragments of 3 kb or longer, thaw one tube of Long Fragment Buffer (LFB) at room temperature, mix by vortexing, spin down and place on ice.

In a 1.5 ml Eppendorf LoBind tube, mix in the following order:

Reagent Volume
Pooled barcoded sample 30 µl
Adapter Mix II T (AMII T) 5 µl
NEBNext Quick Ligation Reaction Buffer (5X) 10 µl
Quick T4 DNA Ligase 5 µl
Total 50 µl

Ensure the components are thoroughly mixed by pipetting, and spin down.

Incubate the reaction for 10 minutes at room temperature.

重要

The next clean-up step uses Long Fragment Buffer (LFB) rather than 70% ethanol to wash the beads. The use of ethanol will be detrimental to the sequencing reaction.

Resuspend the Agencourt AMPure XP beads by vortexing.

Add 20 µl of resuspended Agencourt AMPure XP beads to the reaction and mix by pipetting.

将离心管置于Hula混匀仪（低速旋转式混匀仪）上室温孵育10分钟。

将样品瞬时离心，并静置于磁力架上待磁珠和液相分离。保持离心管在磁力架上不动，用移液枪吸去清液。

Wash the beads by adding 125 μl Long Fragment Buffer (LFB). Flick the beads to resuspend, spin down, then return the tube to the magnetic rack and allow the beads to pellet. Remove the supernatant using a pipette and discard.

重复上述步骤。

将离心管瞬时离心后置于磁力架上。用移液枪吸走残留的上清液。让磁珠在空气中干燥约30秒，但不要干至表面开裂。

Remove the tube from the magnetic rack and resuspend the pellet in 30 µl Elution Buffer (EB).

瞬时离心，然后在37℃下孵育10分钟。请每两分钟轻弹离心管10秒以搅动样本，促进DNA洗脱。

将离心管静置于磁力架上至少一分钟，直到磁珠和液相分离，且洗脱液澄清无色。

Remove and retain 30 µl of eluate containing the DNA library into a clean 1.5 ml Eppendorf DNA LoBind tube.

Dispose of the pelleted beads.

可选操作

Quantify 1 µl of eluted sample using a Qubit fluorometer.

重要

We recommend loading >10 fmols of this final prepared library onto the flow cell for R9.4.1 flow cells.

步骤结束

构建好的文库即可用于测序芯片上样。在上样前，请将文库置于冰上或4℃条件下保存。

提示

文库保存建议

若为 短期 保存或重复使用（例如在清洗芯片后再次上样），我们建议将文库置于Eppendorf LoBind 离心管中 4℃ 保存。 若为一次性使用且储存时长 __超过3个月__，我们建议将文库置于Eppendorf LoBind 离心管中 -80℃ 保存。

可选操作

If quantities allow, the libraries may be diluted in Elution Buffer (EB) for splittling across multiple flow cells.

7. Priming and loading multiple flow cells on a PromethION

材料
  • Flush Buffer (FB)
  • Flush Tether (FLT)
  • Loading Beads II (LBII)
  • Sequencing Buffer II (SBII)
  • Loading Solution (LS)
耗材
  • PromethION 测序芯片
  • 1.5 ml Eppendorf DNA LoBind离心管
  • 2 ml Eppendorf DNA LoBind 离心管
仪器
  • PromethION 2 Solo 测序设备
  • PromethION测序设备
  • PromethION 测序芯片遮光片
  • P1000移液枪和枪头
  • P200 移液枪和枪头
  • P20 移液枪和枪头

Using the Loading Solution

We recommend using the Loading Beads II (LBII) for loading your library onto the flow cell for most sequencing experiments. However, if you have previously used water to load your library, you must use Loading Solution (LS) instead of water. Note: some customers have noticed that viscous libraries can be loaded more easily when not using Loading Beads II.

Thaw the Sequencing Buffer II (SBII), Loading Beads II (LBII), Flush Tether (FLT) and on tube of Flush Buffer (FB) at room temperature before mixing the reagents by vortexing and spin down at room temperature.

重要

Scale up reagent volumes as needed.

Ensure to prepare enough reagents for the total number of flow cells being processed and to take into account extra volume required for pipetting errors.

提示

Each vial provides enough reagent for the preparation of 12 samples. Thaw the appropriate number of vials of each reagent.

Prepare the flow cell priming mix in a suitable vial for the number of flow cells to flush. Once combined, mix well by briefly vortexing.

Reagent Volume per flow cell
Flush Tether (FLT) 30 µl
Flush Buffer (FB) 1,170 µl
重要

将芯片从冰箱中取出后，请将其置于室温环境孵育20分钟再插入PromethION测序仪。潮湿环境下的测序芯片上可能会形成冷凝水。因此，请检查测序芯片顶部和底部的金色连接器引脚处是否有水凝结。如有，请使用无纤维布擦干。请确保测序芯片底部有热垫（黑色）覆盖。

对 PromethION 2 Solo，请按以下步骤为测序芯片上样：

  1. 将测序芯片平放在金属板上。

  2. 将测序芯片推入对接端口，直至金色引脚或绿色电路板不可见。

J2068 FC-into-P2-animation V5

对PromethION 24/48，将测序芯片插入相应卡槽的对接端口：

  1. 将测序芯片与连接器横竖对齐，以便顺利卡入。

  2. 用力下压芯片至卡槽，并确认卡夹位置归位。

Prom Flowcell Loading 1a 中文

Prom Flowcell Loading 1b 中文

重要

如插入配置测试芯片的角度出现偏差，可能会损坏PromethION上的引脚并影响测序结果。如您发现 PromethION测序仪芯片位置上的引脚损坏，请通过电子邮件（support@nanoporetech.com）或微信公众号在线支持（NanoporeSupport）联系我们的技术支持团队。

Screenshot 2021-04-08 at 12.08.37

If not already completed, perform a flow cell check on all flow cells.

Please refer to the Flow Cell Check protocol for further information.

顺时针滑动加液孔孔盖，将其打开。

Prom Flowcell Loading 2 中文

重要

从测序芯片中反旋排出缓冲液。请勿吸出超过20-30µl的缓冲液，并确保芯片上的纳米孔阵列一直有缓冲液覆盖。将气泡引入阵列会对纳米孔造成不可逆转地损害。

在加液孔打开的状态下，按下述步骤吸取少量液体，同时避免引入气泡：

  1. 将P1000移液枪转至200µl刻度。
  2. 将枪头垂直插入加液孔中。
  3. 反向转动移液枪量程调节转纽，直至移液枪刻度在220-230 µl之间，或直至您看到有少量缓冲液进入移液枪枪头。

Prom Flowcell Loading 3 中文

使用P1000移液枪向芯片的加液孔中加入500 µl芯片预处理溶液。加入过程中，请避免引入气泡。等待5分钟，与此同时，您可按以下步骤准备上样文库。

Prom Flowcell Loading 4 中文

Thoroughly mix the contents of the Loading Beads II (LBII) by pipetting.

重要

The Loading Beads II (LBII) tube contains a suspension of beads. These beads settle very quickly. It is vital that they are mixed immediately before use.

In a new tube, prepare the library for loading as follows:

Reagent Volume per flow cell
Sequencing Buffer II (SBII) 75 µl
Loading Beads II (LBII) thoroughly mixed before use, or Loading Solution (LS), if using 51 µl
DNA library 24 µl
Total 150 µl
可选操作

The Multiplex Ligation Kit is designed for users running multiple flow cells. When handling multiple DNA libraries, the Sequencing Buffer (SBII) and Loading Beads II (LBII) can be combined in a master mix:

  1. Mix the Sequencing Buffer II (SBII) and Loading Beads II (LBII) as described above, scaling up the final volume for the appropriate number of samples and adding up to 20% excess of each reagent.
  2. Mix the master mix by pipetting immediately before adding to the DNA samples
  3. Pipette 126 µl of the master mix into each DNA sample-containing tube.
  4. Mix the samples by pipetting.

缓慢向芯片的加液口中加入500 µl预处理液，完成芯片的预处理。

Prom Flowcell Loading 5 中文

临上样前，用移液枪轻轻吹打混匀制备好的文库。

Using a P1000, insert the pipette tip into the inlet port and add 150 µl of library.

Step 6 v2

合上加液孔孔盖。

Prom Flowcell Loading 7 中文

重要

为获得最佳测序产出，在文库样本上样后，请立即在测序芯片上安装遮光片。

我们建议在清洗芯片并重新上样时，将遮光片保留在测序芯片上。一旦文库从测序芯片中吸出，即可取下遮光片。

如遮光片不在测序芯片上，请您按照以下步骤安装：

  1. 将遮光片的中空部分（空槽）与测序芯片的加液孔孔盖对齐。确保遮光片的前沿位于测序芯片ID的上方。
  2. 用力下压遮光片的卡垫部分，遮光片空槽边缘会随卡垫卡入加液孔孔盖下方。

Prom Flowcell Loading 8a 中文

Prom Flowcell Loading 8b 中文

步骤结束

准备就绪后，合上PromethION设备上盖。

请在为PromethION芯片上样后，等待10分钟再启动实验，以提高芯片产出。

For multiple flow cell washing, use the same experiment name and identifying sample IDs for all runs to enable all flow cells to be paused simultaneously.

Screenshot 2023-02-14 114901

8. Data acquisition and basecalling

纳米孔数据分析概览

有关纳米孔数据分析的完整概述，包括碱基识别和次级分析，请参阅 数据分析 文档。

如何开始测序

MinKNOW软件负责仪器控制，数据采集和实时碱基识别。如您已在计算机上安装MinKNOW，则可选择以下几种途径开展测序：

1. 使用计算机上的MinKNOW进行实时数据采集和碱基识别

请按照 MinKNOW 实验指南 的说明操作：从“开始测序”部分起，到“MinKNOW运行结束”部分止。

2. 使用MinION Mk1B/Mk1D测序仪进行实时数据采集和碱基识别

请按照 MinION Mk1B 用户手册 或 MinION Mk1D 用户手册中的说明操作。

3. 使用MinION Mk1C测序仪进行实时数据采集和碱基识别

请参照 MinION Mk1C 用户手册中的说明操作。

4. 使用GridION进行实时数据采集和碱基识别

请参照 GridION 用户手册 中的说明操作。

5. 使用PromethION测序仪进行实时数据采集和碱基识别

请参照 PromethION 用户手册 或 PromethION 2 Solo 用户手册中的说明操作。

6. 使用计算机上的MinKNOW进行数据采集，过后再用NinKNOW进行线下碱基识别

请按照 MinKNOW 实验指南 中的说明操作：从“开始测序”部分起，到“MinKNOW运行结束”部分止。 当您设置实验参数时，请将 碱基识别 选项设为“关”。 测序实验结束后，请按照 MinKNOW 实验指南的本地分析 部分操作。

9. Downstream analysis

Post-basecalling analysis

There are several options for further analysing your basecalled data:

1. EPI2ME platform

The EPI2ME platform is a cloud-based data analysis service developed by Metrichor Ltd., a subsidiary of Oxford Nanopore Technologies. The EPI2ME platform offers a range of analysis workflows, e.g. for metagenomic identification, barcoding, alignment, and structural variant calling. The analysis requires no additional equipment or compute power, and provides an easy-to-interpret report with the results. For instructions on how to run an analysis workflow in EPI2ME, please follow the instructions in the EPI2ME protocol, beginning at the "Starting an EPI2ME workflow" step.

2. Bioinformatics tutorials

For more in-depth data analysis, Oxford Nanopore Technologies offers a range of bioinformatics tutorials, which are available in the Bioinformatics resource section of the Community. The tutorials take the user through installing and running pre-built analysis pipelines, which generate a report with the results. The tutorials are aimed at biologists who would like to analyse data without the help of a dedicated bioinformatician, and who are comfortable using the command line.

3. Research analysis tools

Oxford Nanopore Technologies' Research division has created a number of analysis tools, which are available in the Oxford Nanopore GitHub repository. The tools are aimed at advanced users, and contain instructions for how to install and run the software. They are provided as-is, with minimal support.

4. Community-developed analysis tools

If a data analysis method for your research question is not provided in any of the resources above, please refer to the Community-developed data analysis tool library. Numerous members of the Nanopore Community have developed their own tools and pipelines for analysing nanopore sequencing data, most of which are available on GitHub. Please be aware that these tools are not supported by Oxford Nanopore Technologies, and are not guaranteed to be compatible with the latest chemistry/software configuration.

10. 测序芯片的重复利用及回收

材料
  • 测序芯片清洗剂盒（EXP-WSH004）

完成测序实验后，如您希望再次使用测序芯片，请按照测序芯片清洗试剂盒的说明进行操作，并将清洗后的芯片置于+2至+8℃保存。

您可在纳米孔社区获取 测序芯片清洗试剂盒实验指南。

提示

我们建议您在停止测序实验后尽快清洗测序芯片。如若无法实现，请将芯片留在测序设备上，于下一日清洗。

或者，请按照回收程序将测序芯片返还至Oxford Nanopore。

您可在此处找到回收测序芯片的说明。

重要

如果您遇到问题或对测序实验有疑问，请参阅本实验指南在线版本中的“疑难解答指南”一节。

11. DNA/RNA提取和文库制备过程中可能出现的问题

以下表格列出了常见问题，以及可能的原因和解决方法。

我们还在 Nanopore 社区的“Support”板块 提供了常见问题解答（FAQ）。

如果以下方案仍无法解决您的问题，请通过电邮(support@nanoporetech.com)）或微信公众号在线支持（NanoporeSupport）联系我们。

低质量样本

现象 可能原因 措施及备注
低纯度DNA（Nanodrop测定的DNA吸光度比值260/280<1.8，260/230 <2.0-2.2） 用户所使用的DNA提取方法未能达到所需纯度 您可在 污染物专题技术文档 中查看污染物对后续文库制备和测序实验的影响。请尝试其它不会导致污染物残留的 提取方法。

请考虑将样品再次用磁珠纯化。
RNA完整度低（RNA完整值（RIN）<9.5，或rRNA在电泳凝胶上的条带呈弥散状） RNA在提取过程中降解 请尝试其它 RNA 提取方法。您可在 RNA完整值专题技术文档 中查看更多有关RNA完整值（RIN）的介绍。更多信息，请参阅 DNA/RNA 操作 页面。
RNA的片段长度短于预期 RNA在提取过程中降解 请尝试其它 RNA 提取方法。 您可在 RNA完整值专题技术文档中查看更多有关RNA完整值（RIN）的介绍。更多信息，请参阅DNA/RNA 操作 页面。

我们建议用户在无RNA酶污染的环境中操作，并确保实验设备没有受RNA酶污染.

经AMPure磁珠纯化后的DNA回收率低

现象 可能原因 措施及备注
低回收率 AMPure磁珠量与样品量的比例低于预期，导致DNA因未被捕获而丢失 1. AMPure磁珠的沉降速度很快。因此临加入磁珠至样品前，请确保将磁珠重悬充分混匀。

2. 当AMPure磁珠量与样品量的比值低于0.4:1时，所有的DNA片段都会在纯化过程中丢失。
低回收率 DNA片段短于预期 AMPure磁珠量与样品量的比值越低，针对短片段的筛选就越严格。每次实验时，请先使用琼脂糖凝胶（或其他凝胶电泳方法）确定起始DNA的长度，并据此计算出合适的AMPure磁珠用量。 SPRI cleanup
末端修复后的DNA回收率低 清洗步骤所用乙醇的浓度低于70% 当乙醇浓度低于70%时，DNA会从磁珠上洗脱下来。请确保使用正确浓度的乙醇。

12. Issues during the sequencing run

以下表格列出了常见问题，以及可能的原因和解决方法。

我们还在 Nanopore 社区的“Support”板块 提供了常见问题解答（FAQ）。

如果以下方案仍无法解决您的问题，请通过电邮(support@nanoporetech.com)）或微信公众号在线支持（NanoporeSupport）联系我们。

Mux扫描在测序起始时报告的活性孔数少于芯片质检时报告的活性孔数

现象 可能原因 措施及备注
MinKNOW Mux 扫描在测序起始时报告的活性孔数少于芯片质检时报告的活性孔数 纳米孔阵列中引入了气泡 在对通过质控的芯片进行预处理之前，请务必排出预处理孔附近的气泡。否则，气泡会进入纳米孔阵列对其造成不可逆转地损害。 视频中演示了避免引入气泡的最佳操作方法。
MinKNOW Mux 扫描在测序起始时报告的活性孔数少于芯片质检时报告的活性孔数 测序芯片没有正确插入测序仪 停止测序，将芯片从测序仪中取出，再重新插入测序仪内。请确保测序芯片被牢固地嵌入测序仪中，且达到目标温度。如用户使用的是GridION/PromethION测序仪，也可尝试将芯片插入仪器的其它位置进行测序。
inKNOW Mux 扫描在测序起始时报告的活性孔数少于芯片质检时报告的活性孔数 文库中残留的污染物对纳米孔造成损害或堵塞 在测序芯片质检阶段，我们用芯片储存缓冲液中的质控DNA分子来评估活性纳米孔的数量。而在测序开始时，我们使用DNA文库本身来评估活性纳米孔的数量。因此，活性纳米孔的数量在这两次评估中会有约10%的浮动。

如测序开始时报告的孔数明显降低，则可能是由于文库中的污染物对膜结构造成了损坏或将纳米孔堵塞。用户可能需要使用其它的DNA/RNA提取或纯化方法，以提高起始核酸的纯度。您可在 污染物专题技术文档中查看污染物对测序实验的影响。请尝试其它不会导致污染物残留的 提取方法 。

MinKNOW脚本失败

现象 可能原因 措施及备注
MinKNOW显示 "Script failed”（脚本失败）
重启计算机及MinKNOW。如问题仍未得到解决，请收集 MinKNOW 日志文件 并联系我们的技术支持。 如您没有其他可用的测序设备，我们建议您先将装有文库的测序芯片置于4°C 储存，并联系我们的技术支持团队获取进一步储存上的建议。

Pore occupancy below 40%

Observation Possible cause Comments and actions
Pore occupancy <40% Not enough library was loaded on the flow cell Ensure you load the recommended amount of good quality library in the relevant library prep protocol onto your flow cell. Please quantify the library before loading and calculate mols using tools like the Promega Biomath Calculator, choosing "dsDNA: µg to pmol"
Pore occupancy close to 0 The Ligation Sequencing Kit was used, and sequencing adapters did not ligate to the DNA Make sure to use the NEBNext Quick Ligation Module (E6056) and Oxford Nanopore Technologies Ligation Buffer (LNB, provided in the sequencing kit) at the sequencing adapter ligation step, and use the correct amount of each reagent. A Lambda control library can be prepared to test the integrity of the third-party reagents.
Pore occupancy close to 0 The Ligation Sequencing Kit was used, and ethanol was used instead of LFB or SFB at the wash step after sequencing adapter ligation Ethanol can denature the motor protein on the sequencing adapters. Make sure the LFB or SFB buffer was used after ligation of sequencing adapters.
Pore occupancy close to 0 No tether on the flow cell Tethers are adding during flow cell priming (FLT/FCT tube). Make sure FLT/FCT was added to FB/FCF before priming.

读长短于预期

现象 可能原因 措施及备注
读长短于预期 DNA样本降解 读长反映了起始DNA片段的长度。起始DNA在提取和文库制备过程中均有可能被打断。

1. 1. 请查阅纳米孔社区中的 提取方法 以获得最佳DNA提取方案。

2. 在进行文库制备之前，请先跑电泳，查看起始DNA片段的长度分布。DNA gel2 在上图中，样本1为高分子量DNA，而样本2为降解样本。

3. 在制备文库的过程中，请避免使用吹打或/和涡旋振荡的方式来混合试剂。轻弹或上下颠倒离心管即可。

大量纳米孔处于不可用状态

现象 可能原因 Comments and actions
大量纳米孔处于不可用状态 (在通道面板和纳米孔活动状态图上以蓝色表示)

image2022-3-25 10-43-25 上方的纳米孔活动状态图显示：状态为不可用的纳米孔的比例随着测序进程而不断增加。		 样本中含有污染物 使用MinKNOW中的“Unblocking”（疏通）功能，可对一些污染物进行清除。 如疏通成功，纳米孔的状态会变为"测序孔". 若疏通后，状态为不可用的纳米孔的比例仍然很高甚至增加：

1. 用户可使用 测序芯片冲洗试剂盒（EXP-WSH004）进行核酸酶冲洗 can be performed, 操作，或
2. 使用PCR扩增目标片段，以稀释可能导致问题的污染物。

大量纳米孔处于失活状态

现象 可能原因 措施及备注
大量纳米孔处于失活状态（在通道面板和纳米孔活动状态图上以浅蓝色表示。膜结构或纳米孔遭受不可逆转地损伤） 测序芯片中引入了气泡 在芯片预处理和文库上样过程中引入的气泡会对纳米孔带来不可逆转地损害。请观看 测序芯片的预处理及上样 视频了解最佳操作方法。
大量纳米孔处于失活/不可用状态 文库中存在与DNA共纯化的化合物 与植物基因组DNA相关的多糖通常能与DNA一同纯化出来。

1. 请参考 植物叶片DNA提取方法。
2. 使用QIAGEN PowerClean Pro试剂盒进行纯化。
3. 利用QIAGEN REPLI-g试剂盒对原始gDNA样本进行全基因组扩增。
大量纳米孔处于失活/不可用状态 样本中含有污染物 您可在 污染物专题技术文档 中查看污染物对测序实验的影响。请尝试其它不会导致污染物残留的提取方法。

运行过程中过孔速度和数据质量（Q值）降低

现象 可能原因 措施及备注
运行过程中过孔速度和数据质量（Q值）降低 对试剂盒9系列试剂（如SQK-LSK109），当测序芯片的上样量过多时（请参阅相应实验指南获取推荐文库用量），能量消耗通常会加快。 请按照MinKNOW 实验指南中的说明为测序芯片补充能量。请在后续实验中减少测序芯片的上样量。

温度波动

现象 可能原因 措施及备注
温度波动 测序芯片和仪器接触不良 检查芯片背面的金属板是否有热垫覆盖。重新插入测序芯片，用力向下按压，以确保芯片的连接器引脚与测序仪牢固接触。如问题仍未得到解决，请联系我们的技术支持。

未能达到目标温度

现象 可能原因 措施及备注
MinKNOW显示“未能达到目标温度” 测序仪所处环境低于标准室温，或通风不良（以致芯片过热） MinKNOW会限定测序芯片达到目标温度的时间。当超过限定时间后，系统会显示出错信息，但测序实验仍会继续。值得注意的是，在错误温度下测序可能会导致通量和数据质量（Q值）降低。请调整测序仪的摆放位置，确保其置于室温下、通风良好的环境中后，再在MinKNOW中继续实验。有关MinION温度控制的更多信息，请参考此 FAQ （常见问题）文档。

Guppy – no input .fast5 was found or basecalled

Observation Possible cause Comments and actions
No input .fast5 was found or basecalled input_path did not point to the .fast5 file location The --input_path has to be followed by the full file path to the .fast5 files to be basecalled, and the location has to be accessible either locally or remotely through SSH.
No input .fast5 was found or basecalled The .fast5 files were in a subfolder at the input_path location To allow Guppy to look into subfolders, add the --recursive flag to the command

Guppy – no Pass or Fail folders were generated after basecalling

Observation Possible cause Comments and actions
No Pass or Fail folders were generated after basecalling The --qscore_filtering flag was not included in the command The --qscore_filtering flag enables filtering of reads into Pass and Fail folders inside the output folder, based on their strand q-score. When performing live basecalling in MinKNOW, a q-score of 7 (corresponding to a basecall accuracy of ~80%) is used to separate reads into Pass and Fail folders.

Guppy – unusually slow processing on a GPU computer

Observation Possible cause Comments and actions
Unusually slow processing on a GPU computer The --device flag wasn't included in the command The --device flag specifies a GPU device to use for accelerate basecalling. If not included in the command, GPU will not be used. GPUs are counted from zero. An example is --device cuda:0 cuda:1, when 2 GPUs are specified to use by the Guppy command.

Last updated: 12/6/2023

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