本实验指南详细描述了使用连接测序试剂盒V14（SQK-LSK114）进行DNA样品测序的操作流程及常见问题的解决方法。为使您尽快熟悉操作流程，我们强烈建议您在使用样品测序前，先使用Lambda标准品完成标准对照实验。

有关双链碱基识别的信息，请参阅 Kit 14 测序和互补双链碱基识别 信息技术表。

测序工作流程：

实验准备

您将需要：

• 提取DNA，并评估DNA的长度、浓度和纯度 质量评估步骤对确保实验成功至关重要。
• 确保您已准备好测序试剂盒、正确的仪器以及第三方试剂
• 下载数据收集和分析软件
• 检查您的测序芯片上有足够多的活性纳米孔，以确保测序良好运行

文库制备

下表概述了文库制备所需的步骤，包括时间安排和可以中止的节点。

文库制备	步骤	时间	中止节点
DNA损伤及末端修复	修复DNA，并对DNA进行末端修复以便与接头连接	35 分钟	4°C 过夜
接头连接及纯化	将测序接头连接到DNA末端	20 分钟	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-80℃保存。 我们强烈建议您在为文库连接接头后，尽快测序。
测序芯片预处理及上样	对测序芯片进行预处理，然后将DNA文库加至芯片中进行测序。	5 分钟

测序和分析

您将需要：

• 使用 MinKNOW 软件运行测序，该软件将收集由测序仪产出的原始数据并将其识别为碱基序列。

• 使用EPI2ME软件中的生物信息学工作流程分析您的数据。

Input requirements per sample for the extraction method:

• ≥ 1 ml of human blood in EDTA K2 vacuum tube

Input requirements per sample for the library preparation:

• 3 µg of SFE size selected and Megaruptor fragmented gDNA

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。

Oxford Nanopore Technologies推荐您使用本实验指南中提及的所有第三方试剂，并已对其加以验证。我们尚未对其它替代试剂进行测试。

我们建议您按制造商说明准备待用的第三方试剂.

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。

请注意： 我们正在将部分试剂的包装形式由单次管装改为瓶装。

单次管装试剂：

部分试剂改为瓶装：

声明： 本产品包含由贝克曼库尔特公司（Beckman Coulter, Inc）生产的 AMPure XP 试剂，并可与试剂盒一起于-20°C 下储存（试剂稳定性将不受损害）。

请注意： DNA参照（DCS）是一段可比对到Lambda基因组的3'端、长度为3.6 kb 的标准扩增子。

Overlay of fragment length profiles of a) gDNA extracted using the Puregene Blood Kit, b) gDNA extracted and size selected using SFE buffer. c) gDNA extracted, size selected and fragmented using Megaruptor 3 (MR).

For this method, the flow cell is washed after ~22 hours of sequencing to restore pores to ensure efficient data acquisition. After an additional 22 hours of sequencing, the flow cell is washed and reloaded a second time. For this reason, enough library was generated for 3 flow cell loads in the adapter ligation step of the protocol.

• This washing procedure aims to remove most of the initial library and unblock the pores to prepare the flow cell for the loading of a subsequent library.
• Data acquisition in MinKNOW should be paused during the wash procedure and library loading.
• After the flow cell has been washed, the next library can be loaded.

You can navigate to the Pore Activity or the Pore Scan Results plot to see pore availability.

Below you can find example data for pore states observed on a flow cell before and after wash steps are performed. Additionally, you can observe an example for the cummulative sequencing data output, including the wash and reload steps. The red asterisks indicate the flow cell wash and reloads.

Figure 1. Channel state over a 72 hour run. The flow cell washes are incorporated into the method to restore blocked pores, to allow continuous data acquisition. Red asterisks denote when a flush was performed.

Figure 2. Cumulative sequencing data output, over a 72 hour run. Red asterisks denote when a flush was performed.

Figure 3. Read length profile for a 30 kb N50 library. The approximately gaussian shape is characteristic of gDNA that has undergone SFE size selection and Megaruptor shearing. The short distribution of reads at ~5kb is due to premature termination of reads at the mux scan.

The sequencing device control, data acquisition and real-time basecalling are carried out by the MinKNOW software.

We recommend basecalling with the high accuracy (HAC) basecaller in real-time with BAM selected as output type using the P2 Solo or P24/P48 device.

You must generate a BAM file from your sequening run, as this is required for input into the wf-human-variation workflow.

Refer to the links below containing the detailed instructions for setting up the device and sequencing run:

• PromethION 24 and 48: "Starting a sequencing run with PromethION 24 and 48"

• PromethION 2 Solo: "Starting a sequencing run on PromethION 2 Solo"

Below are the recommended sequencing parameters for MinKNOW.

We recommend setting the run time to 100 hours to accommodate for the two flow cell washes, using the modified bases option for basecalling and ensuring a BAM output is selected. All other sequencing parameters can be kept to their default settings. Below are our current recommendations:

Positions

Flow cell position: [user defined]

Experiment name: [user defined]

Flow cell type: FLO-PRO114M

Sample ID: [user defined]

Kit

Kit selection: Ligation Sequencing Kit (SQK-LSK114)

Run configuration

Sequencing and analysis

Basecalling: On [default] Modified bases: On with '5mC & 5hmC CG contexts' selected Model: High-accuracy basecalling (HAC) [default]

Barcoding: Disabled [default]

Alignment: Off [default]
We do not currently recommend live alignment during sequencing, as it can slow down system processing.

Adaptive sampling: Off [default]

Advanced options Active channel selection: On [default] Time between pore scans: 1.5 [default] Reserve pores: On [default]

Data targets

Run limit: 100 hours [default]

Output

Output format .POD5: On [default] .FASTQ: On [default] .BAM: On

Filtering: On [default] Qscore: 9 [default] Minimum read length: 200 bp [default]

For the analysis of human blood DNA sequence data, we recommend the wf-human-variation workflow. This end-to-end software pipeline is implemented using the Nextflow workflow language and implements methods for the calling of single nucleotide polymorphisms (SNPs), structural variants (SVs), and for reporting DNA methylation information.

The wf-human-variation workflow is best run from the BAM file produced by MinKNOW when the modified base model for basecalling is selected. If sequence read mapping to the reference genome is not performed by MinKNOW, we recommend to perform the basecalling using the wf-basecalling workflow. Ensure you save the outputs in BAM format by providing the --output_bam option.

The tools below are used in the analysis workflow and can be run in isolation or together:

1. Sniffles2 calls SVs and file output include an HTML report of QC metrics and VCF format list of variants and their quality scores.

2. Clair3 calls SNPs and file output includes an HTML report of QC metrics and VCF format list of variants and their quality scores.

3. modkit extracts methylation information from the provided BAM file which is summarised in a BEDmethyl format file.

The wf-human-variation workflow is preconfigured using appropriate parameters and requires tuning only for the choice of reference genome and Clair3 model. Please see the project’s documentation for further details.

The results from the wf-human-variation workflow can be further explored by viewing in a track-based genome browser such as IGV can be assessed for known pathogenicity through tertiary analysis software.

The wf-human-variation workflow is intended to be run using the Nextflow software.

We recommend performing downstream analysis using EPI2ME which facilitates bioinformatic analyses by allowing users to run Nextflow workflows in a Graphical User Interfaces (GUI). EPI2ME maintains a collection of bioinformatic workflows which are curated and actively maintained by experts in long-read sequence analysis. The collection of all our available EPI2ME workflows can be found here.

For new users, the quick start guide can be found here outlining how to use this interface.

The primary workflow outputs include:

• gzipped VCF file containing the SNPs in the dataset from --snp
• gzipped VCF file containing the SVs in the dataset from --sv
• gzipped bedMethyl file aggregating modified base counts from --mod
• HTML report detailing the primary findings of the workflow for QC metrics, and SNP and SV calling
• If an unaligned BAM file was provided, the workflow will ouput a CRAM file containing the alignments used to make the downstream variant calls.

The secondary workflow outputs:

• mosdepth ouputs include:
  • {sample_name}.mosdepth.global.dist.txt: a cumulative distribution indicating the proportion of total bases for each and all reference sequences
    
  • {sample_name}.regions.bed.gz: the mean coverage for each region in the provided BED file
    
  • {sample_name}.thresholds.bed.gz: the number of bases in each region that are covered at or above each threshold value (1, 10, 20, 30X)

• bamstats ouputs include:
  • {sample_name}.readstats.tsv.gz: a gzipped TSV summarising per-alignment statistics produced by bamstats
  • {sample_name}.ftagstat.tsv: a text file with summary alignment statistics for each reference sequence

It is possible to phase SNPs, SVs and modified bases by providing the --phased option.

To improve the accuracy of SV calling, specify a suitable tandem repeat BED for your reference with --tr_bed.

Aggregation of methylation calls with --mod requires data to be basecalled with a model that includes base modifications, providing the MM and ML BAM tags. To do so on MinKNOW, ensure 'Modified bases' option is selected during basecalling set up, with the '5mC' model selected.

Ensure to retain the input reference when basecalling or alignment is performed as CRAM files cannot be read without the corresponding input reference.

For a full list of available basecalling models, refer to the Dorado documentation.

Due to the extended sequencing time, and the multiple flow cell washes and library reloads, we do not recommend re-using the flow cells used in this method.

Re-using these flow cells for subsequent sequencing experiments may result in insufficient data generation for analysis.

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。

Observation	Possible cause	Comments and actions
Low DNA purity (Nanodrop reading for DNA OD 260/280 is <1.8 and OD 260/230 is <2.0–2.2)	The DNA extraction method does not provide the required purity	The effects of contaminants are shown in the Contaminants Know-how piece. Please try an alternative extraction method that does not result in contaminant carryover. Consider performing an additional AMPure bead clean-up step.

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。
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。

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。

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 。

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。

Observation	Possible cause	Comments and actions
Pore occupancy <40%	Not enough library was loaded on the flow cell	For the human genome sequencing protocols, 200-300 ng of good quality library should be loaded on to an R10.4.1 flow cell to keep pore occupancy high.
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 SQK-LSK114 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 (FCT tube). Make sure FCT was added to FCF before priming.

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。 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。

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) 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。

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。

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。

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 MK1B温度控制的更多信息，请参考此 FAQ （常见问题）文档。