Direct-from-colony microbial sequencing: rapid Salmonella serotyping (SQK-RPB114.24)

Oxford Nanopore Technologies
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MinION: Protocol

Direct-from-colony microbial sequencing: rapid Salmonella serotyping (SQK-RPB114.24) V DCM_9210_V114_revB_30Sep2024

This protocol:

  • is a rapid method to perform sequencing and analysis of Salmonella.
  • uses Salmonella colonies from overnight cultures.
  • includes tagmentation, barcoding and PCR amplification.
  • enables multiplexing of 1-24 samples.
  • is compatible with R10.4.1 flow cells.

For Research Use Only

This is an Early Access product For more information about our Early Access programmes, please see this article on product release phases.

FOR RESEARCH USE ONLY

概览

This protocol:

  • is a rapid method to perform sequencing and analysis of Salmonella.
  • uses Salmonella colonies from overnight cultures.
  • includes tagmentation, barcoding and PCR amplification.
  • enables multiplexing of 1-24 samples.
  • is compatible with R10.4.1 flow cells.

For Research Use Only

This is an Early Access product For more information about our Early Access programmes, please see this article on product release phases.

Document version: DCM_9210_V114_revB_30Sep2024

1. Overview of the protocol

重要

本试剂盒为早期试用产品

如需有关早期试用计划的更多信息，请参阅 本文了解产品的不同发布阶段。

请确保您始终使用最新版本的实验指南。

Introduction to the direct-from-colony microbial sequencing: rapid Salmonella serotyping protocol

This protocol describes a method for preparing, sequencing and analysis of up to 24 Salmonella colonies using the Rapid RCR Barcoding Kit V14 (SQK-RPB114.24).

This simple end-to-end solution allows you to generate whole genome sequencing data directly from a single Salmonella colony, eliminating the need for subculture into liquid broth, DNA extraction or complex library preparation methods, thereby offering faster tun-around times.

Combined with our custom wf-bacterial-genomes analysis pipeline using EPI2ME, this method provides an organism species ID, serotype, multi locus sequence type (MLST) and antibiotic resistance (AMR) profile information.

This method is designed to provide non-circular genomes for research and routine lab operations, including food safety testing purposes.

For more information and additional options for this end-to-end workflow please visit our know-how document.

Salmonella serotyping RPB114 workflow

Steps in the end-to-end workflow:

Prepare for your experiment

You will need to:

  • Ensure pathogenic organisms are handled in appropriate biosafety conditions. Please adhere to the correct health and safety practices in accordance to your laboratory standards and local rules and regulations.
    This method should be performed in the appropriate containment level laboratory spaces (biosafety cabinets: BSC) until the sample tagmentation stage. Following sample tagmentation, cells are non-viable and can be used outside the biosafety cabinet.
  • Streak your sample onto a freshly prepared agar plate appropriately such that single Salmonella colonies can be isolated (overnight). We recommend the use of TSA plates for culturing Salmonella.

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

Library preparation

The table below is an overview of the steps required in the library preparation, including timings and optional stopping points.

Step Process Time Stop option
Colony growth Grow Salmonella single colony culture(s) of ~2-3 mm overnight on an agar plate. ~16-18 hours
Sample preparation Resuspend each single colony in Tris Buffer. 5 minutes
DNA tagmentation Tagment your DNA using the Fragmentation Mix from the sequencing kit 10 minutes
PCR amplification PCR your sample using the barcoded primer supplied in the sequencing kit 180 minutes We do not recommend leaving the PCR reaction overnight, using the amplified samples straight after PCR will yield better results.

If sample storage is unavoidable, add 4 uL EDTA to each sample and then stored at 4°C.
Sample pooling and cleanup Pool your barcoded PCR samples in equal ratios and perform a clean-up 30 minutes 4°C overnight
Adapter ligation and clean-up Attach the sequencing adapters to the DNA ends 5 minutes We strongly recommend sequencing your library as soon as it is adapted.
Priming and loading the flow cell Prime the flow cell and load the prepared library for sequencing 5 minutes

Sequencing and analysis

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
  • Start the EPI2ME software and select the wf-bacterial-genomes workflow
重要

Compatibility of this protocol

This protocol should only be used in combination with:

2. Equipment and consumables

材料
  • Up to 24x single Salmonella colonies (2–3 mm in diameter)
  • Rapid PCR Barcoding Kit 24 V14 (SQK-RPB114.24)
耗材
  • MinION及GridION测序芯片
  • 1 M Tris Buffer, pH8 (e.g. Invitrogen™, AM9855G)
  • LongAmp Hot Start Taq 2X Master Mix (NEB, M0533)
  • 无核酸酶水（如ThermoFisher，AM9937）
  • 新制备的80%乙醇（用无核酸酶水配制）
  • （非必需）牛血清白蛋白（BSA）（50 mg/mL）（例如 Invitrogen™ UltraPure™ BSA （50 mg/mL）， AM2616）
  • Qubit dsDNA HS Assay（双链DNA高灵敏度检测）试剂盒（Invitrogen, Q32851）
  • Qubit™ 分析管（Invitrogen, Q32856）
  • 1.5 ml Eppendorf DNA LoBind 离心管
  • 0.2 ml 薄壁PCR管
仪器
  • MinION 或 GridION 测序仪
  • MinION 及GridION 测序芯片遮光片
  • Inoculation loop or equivalent for picking your colony
  • 盛有冰的冰桶
  • 迷你离心机
  • 计时器
  • 热循环仪
  • 磁力架
  • Hula混匀仪（低速旋转式混匀仪）
  • 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
  • P2 移液枪和枪头
  • 多通道移液枪和枪头
  • 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）
  • Microbiological Safety Cabinet II contamination level 3

For this protocol, you will need a single colony of Salmonella 2–3 mm in diameter for each input.

Streak your sample onto a freshly prepared agar plate appropriately such that single Salmonella colonies can be isolated (overnight). We recommend the use of TSA plates for culturing Salmonella.

Optional: Alternatively, you can use 3-5 ng in 3 μl volume per sample of isolated DNA as input for library preparation.

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。

第三方试剂

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.

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。

重要

The Rapid Adapter (RA) used in this kit and protocol is not interchangeable with other sequencing adapters.

Rapid PCR Barcoding Kit 24 V14 (SQK-RPB114.24) contents

sqk-rpb114.24 tubes

Name Acronym Cap colour No. of vials Fill volume per vial (µl)
Fragmentation Mix FRM Brown 1 160
Rapid Adapter RA Green 1 15
Adapter Buffer ADB Clear 1 100
AMPure XP Beads AXP Amber 3 1,200
Elution Buffer EB Black 2 500
EDTA EDTA Blue 1 700
Sequencing Buffer SB Red 1 700
Library Beads LIB Pink 1 600
Library Solution LS White cap, pink label 1 600
Flow Cell Flush FCF Clear 1 8,000
Flow Cell Tether FCT Purple 1 200
Rapid Barcode Primer 01-24 RLB01-24 Clear 24 (one per barcode) 15

Note: This product contains AMPure XP Reagent manufactured by Beckman Coulter, Inc. and can be stored at -20°C with the kit without detriment to reagent stability.

Rapid barcode primers

Component Sequence
RLB01 AAGAAAGTTGTCGGTGTCTTTGTG
RLB02 TCGATTCCGTTTGTAGTCGTCTGT
RLB03 GAGTCTTGTGTCCCAGTTACCAGG
RLB04 TTCGGATTCTATCGTGTTTCCCTA
RLB05 CTTGTCCAGGGTTTGTGTAACCTT
RLB06 TTCTCGCAAAGGCAGAAAGTAGTC
RLB07 GTGTTACCGTGGGAATGAATCCTT
RLB08 TTCAGGGAACAAACCAAGTTACGT
RLB09 AACTAGGCACAGCGAGTCTTGGTT
RLB10 AAGCGTTGAAACCTTTGTCCTCTC
RLB11 GTTTCATCTATCGGAGGGAATGGA
RLB12 GTTGAGTTACAAAGCACCGATCAG
RLB13 AGAACGACTTCCATACTCGTGTGA
RLB14 AACGAGTCTCTTGGGACCCATAGA
RLB15 AGGTCTACCTCGCTAACACCACTG
RLB16 CGTCAACTGACAGTGGTTCGTACT
RLB17 ACCCTCCAGGAAAGTACCTCTGAT
RLB18 CCAAACCCAACAACCTAGATAGGC
RLB19 GTTCCTCGTGCAGTGTCAAGAGAT
RLB20 TTGCGTCCTGTTACGAGAACTCAT
RLB21 GAGCCTCTCATTGTCCGTTCTCTA
RLB22 ACCACTGCCATGTATCAAAGTACG
RLB23 CTTACTACCCAGTGAACCTCCTCG
RLB24 GCATAGTTCTGCATGATGGGTTAG

3. Library preparation

材料
  • Up to 24x single Salmonella colonies (2–3 mm in diameter)
  • Fragmentation Mix (FRM)
  • Rapid Barcode Primers (RLB01-24, at 10 µM)
  • EDTA（EDTA）
  • AMPure XP 磁珠（AXP）
  • Elution Buffer (EB)
  • 快速测序文库接头（RA）
  • 接头缓冲液（ADB）
耗材
  • 1 M Tris Buffer, pH8 (e.g. Invitrogen™, AM9855G)
  • LongAmp Hot Start Taq 2X Master Mix (NEB, M0533)
  • 无核酸酶水（如ThermoFisher，AM9937）
  • 新制备的80%乙醇（用无核酸酶水配制）
  • Qubit dsDNA HS Assay（双链DNA高灵敏度检测）试剂盒（Invitrogen, Q32851）
  • Qubit™ 分析管（Invitrogen, Q32856）
  • 1.5 ml Eppendorf DNA LoBind 离心管
  • 0.2 ml 薄壁PCR管
仪器
  • Inoculation loop or equivalent for picking your colony
  • 盛有冰的冰桶
  • 热循环仪
  • Hula混匀仪（低速旋转式混匀仪）
  • 磁力架
  • 迷你离心机
  • 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
可选仪器
  • Microbiological Safety Cabinet II contamination level 3
注意

Ensure pathogenic organisms and samples are handled in appropriate biosafety conditions.

Please adhere to the correct health and safety practices in accordance to your laboratory standards and local rules and regulations.

This method should be performed in the appropriate containment level laboratory spaces (biosafety cabinets: BSC) until the sample tagmentation stage. Following sample tagmentation, cells are non-viable and can be used outside the biosafety cabinet.

Thaw kit components at room temperature, spin down briefly using a microfuge and mix by pipetting as indicated by the table below:

Reagent 1. Thaw at room temperature 2. Briefly spin down 3. Mix well by pipetting or vortexing
Rapid Barcode Primers (RLB01-24) Not frozen ✓ Pipette
Fragmentation Mix (FRM) Not frozen ✓ Pipette
Rapid Adapter (RA) Not frozen ✓ Pipette
Adapter Buffer (ADB) ✓ ✓ Vortex or Pipette
AMPure XP Beads (AXP) ✓ ✓ Mix by pipetting or vortexing immediately before use
Elution Buffer (EB) ✓ ✓ Vortex or Pipette
EDTA (EDTA) ✓ ✓ Vortex or Pipette

Note: Once thawed, keep all kit components on ice.

重要

When picking a single colony, please ensure that no media is carried through as this can negatively impact the PCR yields.

Prepare each cultured cell colony as follows:

  • For each colony/sample, add 50 µl 10 mM tris buffer (pH 8) to a 1.5 ml Eppendorf DNA LoBind tube.
  • Using an inoculation loop, transfer a single 2–3 mm colony from an overnight culture into a separate tube (from the step above).
  • Resuspend each colony thoroughly by pipette mixing and briefly vortexing.
  • Ensure your cell colony suspension is homogenous before proceeding.

In a separate 0.2 ml thin-walled PCR tube for each sample, mix the following:

Reagent Volume
DNA/colony suspension (from previous step) 3 μl
Fragmentation Mix (FRM) 1 μl
Total 4 μl

Mix thoroughly by pipetting the reaction.

In a thermal cycler, incubate the tube at 30°C for 2 minutes, then at 80°C for 2 minutes.

Sample tagmentation has taken place, cells are now non-viable and can be used outside the biosafety cabinet.

Please continue to adhere to the correct health and safety practices in accordance to your laboratory standards and local rules and regulations.

For each sample, set up a PCR reaction as follows in the 0.2 ml thin-walled PCR tube containing the tagmented DNA:

Reagent Volume
Tagmented DNA (from previous step) 4 µl
Nuclease-free water 20 µl
RLB (01-24, at 10 µM) 1 µl
LongAmp Hot Start Taq 2X Master Mix 25 µl
Total 50 µl

If the amount of input material is altered, the number of PCR cycles may need to be adjusted to produce the same yield.

轻弹离心管以充分混合，并瞬时离心。

Amplify using the following cycling conditions:

Cycle step Temperature Time No. of cycles
Initial denaturation 95°C 3 mins 1
Denaturation

Annealing

Extension		 95°C

56°C

65°C		 15 sec

15 sec

6 min

25*
Final extension 65°C 6 min 1
Hold 4°C ∞

For more information on the PCR cycle number and externsion times please visit our know-how document.

Add 4 µl of EDTA to each barcoded sample, mix thorougly by pipetting and spin down briefly.

提示

在此步骤中添加EDTA的目的是终止反应。

Incubate for 5 minutes at room temperature.

Quantify 1 µl of each barcoded sample using a Qubit fluorometer (or equivalent) for QC check.

Note: The expected PCR yield for each sample is 15–45 ng/µl.

Pool 100 ng of each PCR barcoded samples in a clean 1.5 ml Eppendorf DNA LoBind tube.

Note: Please ensure you have quantified your samples prior to this step and take forward 100 ng of each of the samples for optimal barcode balancing.
Samples may vary in concentration following the barcoded PCR, therefore the volume of each barcoded sample added to the pool will be different.

Make up the volume of pooled samples to the nearest 100 µl using nuclease free water.

For example:

  • if the 100 ng pool of each sample results in a total of 88 µl, add 12 µl of nuclease-free water to make up the volume to 100 µl.
  • if the 100 ng pool of each sample results in a total of 180 µl, add 20 µl of nuclease-free water to make up the volume to 200 µl.

Resuspend the AMPure XP Beads (AXP) by vortexing.

To the pool of barcoded samples, add a 0.6X volume ratio of resuspended AMPure XP Beads (AXP) and mix by pipetting:

Volume of barcoded sample pool 100 μl 200 μl 300 μl
Volume of AMPure XP Beads (AXP) 60 μl 120 μl 180 μl

Incubate the reaction on a hula mixer (rotator mixer) for 10 minutes at room temperature.

准备 2 ml 新制备的80％乙醇（用无核酸酶水配制）。

Briefly spin down the sample and pellet on a magnetic rack until supernatant is clear and colourless. Keep the tube on the magnetic rack, and pipette off the supernatant.

Keep the tube on the magnet and wash the beads with 1 ml of freshly-prepared 80% 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。

Remove the tube from the magnetic rack and resuspend the pellet by pipetting in 15 µl Elution Buffer (EB). Spin down and incubate for 5 minutes at room temperature.

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 15 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.

  • Remove and retain the eluate which contains the DNA library in a clean 1.5 ml Eppendorf DNA LoBind tube
  • Dispose of the pelleted beads
CHECKPOINT

Quantify 1 µl of eluted sample using a Qubit fluorometer.

Transfer 250 ng of your eluted samples into a clean 1.5 ml Eppendorf DNA LoBind tube. Make up the volume to 11 µl with Elution Buffer (EB).

Note: If your final yield is below 250 ng, take forward 11 µl of eluted sample for the maxium flow cell loading amount possible.

Please note, if you underload the flow cell you might need to sequence for longer than stated in this method to achieve the recommended number of reads (at least 50k reads per barcode).

在一支1.5ml Eppendorf DNA LoBind离心管内，按下表稀释快速测序文库接头（RA），并吹打混匀：

试剂 体积
快速测序文库接头（RA） 1.5 μl
接头缓冲液（ADB） 3.5 μl
总体积 5 μl

向11 μl带条形码的DNA洗脱液中加入1 μl 经过稀释的快速测序文库接头（RA）。

轻弹离心管以充分混合，并瞬时离心。

Incubate the reaction for 5 minutes at room temperature.

步骤结束

制备好的文库即可用于芯片上样。请在上样前，始终将文库置于冰上。

4. MinION 及 GridION 测序芯片的预处理及上样 (1)

材料
  • 测序芯片冲洗液（FCF）
  • 测序芯片系绳（FCT）
  • 文库颗粒（LIB）
  • 测序缓冲液（SB）
耗材
  • MinION及GridION测序芯片
  • （非必需）牛血清白蛋白（BSA）（50 mg/mL）（例如 Invitrogen™ UltraPure™ BSA （50 mg/mL）， AM2616）
  • 1.5 ml Eppendorf DNA LoBind 离心管
仪器
  • MinION 或 GridION 测序仪
  • MinION 及GridION 测序芯片遮光片
  • P1000 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
重要

请注意：本试剂盒仅兼容R10.4.1测序芯片（FLO-MIN114）。

提示

测序芯片的预处理及上样

我们建议所有新用户在首次运行测序芯片前，观看视频测序芯片的预处理及上样。

于室温下解冻测序缓冲液（SB）、文库颗粒（LIB）或文库溶液（LIS）、测序芯片系绳（FCT）和一管测序芯片冲洗液（FCF）。完全解冻后，涡旋振荡混匀，然后瞬时离心并置于冰上。 (1)

重要

为在MinION及GridION R10.4.1测序芯片（FLO-MIN114）上获得最优的测序表现并提高测序产出，我们推荐您向测序芯片预处理液中加入终浓度为0.2 mg/ml的牛血清白蛋白（BSA）。

请注意： 我们不推荐使用其它类型的白蛋白（例如重组人血清白蛋白）。

在一支洁净的 1.5 ml Eppendorf DNA LoBind离心管中，按下表制备测序芯片的预处理液，室温下颠倒离心管并吹打混匀：

试剂 体积（每张芯片）
测序芯片冲洗液 (FCF) 1170 µl
50mg/ml的牛血清白蛋白 (BSA) 5 µl
测序芯片系绳 (FCT) 30 µl
总体积 1205 µl

打开MinION或GridION测序仪的盖子，将测序芯片插入金属固定夹的下方。用力向下按压芯片，以确保正确的热、电接触。

中文-测序芯片预处理上样1a

中文-测序芯片预处理上样1b

可选操作

为文库上样前，完成测序芯片检测，查看可用孔数目。

如此前已对测序芯片进行过质检，则此步骤可省略。

更多信息，请查看MinKNOW实验手册的 测序芯片质检 部分。

顺时针转动预处理孔孔盖，使预处理孔显露出来。

中文-测序芯片预处理上样2

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。

将预处理孔打开后，检查孔周围是否有小气泡。请按照以下方法，从孔中排出少量液体以清除气泡：

  1. 将P1000移液枪转至200µl刻度。
  2. 将枪头垂直插入预处理孔中。
  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。
    __请注意：__ 肉眼检查，确保从预处理孔到传感器阵列的缓冲液连续且无气泡。

中文-测序芯片预处理上样3

通过预处理孔向芯片中加入800µl预处理液，避免引入气泡。等待5分钟。在此期间，请按照以下步骤准备用于上样的DNA文库。

中文-测序芯片预处理上样4

将含有文库颗粒的LIB管用移液枪吹打混匀。

重要

LIB管内的文库颗粒分散于悬浮液中。由于颗粒沉降速度非常快，因此请在混匀颗粒后立即使用。

对于大多数测序实验，我们建议您使用文库颗粒（LIB）。但如文库较为粘稠，您可考虑使用文库溶液（LIS）。

在一支新的1.5ml Eppendorf LoBind离心管中，按下表所示准备上样文库： (1)

试剂 体积（每张测序芯片）
测序缓冲液（SB） 37.5 µl
文库颗粒（LIB），使用前即时混匀；或文库溶液（LIS） 25.5 µl
DNA文库 12 µl
总体积 75 µl

完成测序芯片的预处理：

  1. 轻轻地翻起SpotON上样孔盖，使SpotON上样孔显露出来。 中文-测序芯片预处理上样5
  2. 通过预处理孔（而 非 SpotON加样孔）向芯片中加入200µl预处理液，避免引入气泡。 中文-测序芯片预处理上样6

临上样前，用移液枪轻轻吹打混匀制备好的文库。

通过SpotON加样孔向芯片中逐滴加入75µl样品。确保液滴流入孔内后，再加下一滴。

中文-测序芯片预处理上样7

轻轻合上SpotON加样孔孔盖，确保塞头塞入加样孔内。逆时针转动预处理孔孔盖，盖上预处理孔。

中文-测序芯片预处理上样8

中文-测序芯片预处理上样9

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。

按下述步骤安装测序芯片遮光片：

  1. 小心将遮光片的前沿（平端）与金属固定夹的边沿对齐。 请注意： 请勿将遮光片强行压到固定夹下方。

  2. 将遮光片轻轻盖在测序芯片上。遮光片的SpotON加样孔孔盖缺口应与芯片上的SpotON加样孔孔盖接合，遮盖住整个测序芯片的前部。

MinION加装遮光片

注意

MinION测序芯片的遮光片并非固定在测序芯片上，因此当为芯片加装遮光片后，请小心操作。

步骤结束

小心合上测序设备上盖并在MinKNOW上设置测序实验。

5. 数据采集和碱基识别 (1)

如何开始测序 (1)

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：从“开始测序”部分起，到“MinKNOW运行结束”部分止。

2. 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。

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 使用指南中的说明。

4. 使用PromethION测序仪进行实时数据采集和碱基识别

请参照 PromethION 使用指南 或 PromethION 2 Solo 使用指南中的说明。

5. 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：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。

MinKNOW settings for Salmonella serotyping

For fastest turnaround time and easiest analysis, basecalling should be performed live during the sequencing run using the High Accuracy (HAC) basecaller.

Below are the recommendations for MinKNOW settings:

Positions

Flow cell position: [user defined]

Experiment name: [user defined]

Flow cell type: FLO-MIN114

Sample ID: [user defined]

Kit

Kit selection: Rapid PCR Barcoding Kit (SQK-RPB114.24)

Run configuration

Sequencing and analysis

Basecalling: On [default] Modified bases: Off Model: High-accuracy basecalling (HAC) [default]

Barcoding: On [default] Trim barcodes: Off [default] Barcode both ends: Off [default] Custom barcodes selection: Off [default]

Alignment: Off [default]

Adaptive sampling: Off [default]

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

Data targets

Sequencing time will vary according to the number of samples being sequenced. The aim is to generate 50k reads per sample/barcode.

Run limit: Set according to number of sampeles:

Number of samples/barcodes Recommended sequencing time
6 4 hours
12 8 hours
24 12 hours

Output

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

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

6. Downstream analysis

Post-basecalling analysis

We recommend performing downstream analysis using EPI2ME which facilitates bioinformatic analyses by allowing users to run Nextflow workflows in a desktop application. EPI2ME maintains a collection of bioinformatic workflows which are curated and actively maintained by experts in long-read sequence analysis.

Further information about the available EPI2ME workflows are available here, along with the Quick Start Guide to start your first bioinformatic workflow.

For the accurate and reliable assembly or alignment of you sample genomes, we recommend using the wf-bacterial-genomes workflow. At its core, the workflow is an efficient assembly pipeline which also polishes genomes using Medaka.

Whilst running the workflow using the default parameters will , using the ‘Isolates’ mode to produce high quality genome assemblies, perform additional analyses designed to increase genome quality and aid genome interrogation for common pathogens in the clinical and food safety fields. ‘Isolates’ analyses includes MLST(7-gene), species confirmation and AMR prediction in addition to sample-specific reports.

Note: You can also run this workflow through command line. However, we only recommend this option for experienced users. For more information, please visit the wf-bacterial-genomes page on GitHub and the know-how document.

重要

Please ensure your device meets the minimum compute requirements to run this workflow.

Compute requirements:

CPUs Memory
Minimum requirements: 8 32GB
Recommended requirements: 16 64GB

For more information visit the EPI2ME wf-bacterial-genomes documentation.

Open the EPI2ME app using the desktop shortcut.

On the landing page, open the workflow tab on the left-hand sidebar.

1 landing page EPI2ME

Navigate to the Available workflows tab and click on wf-bacterial-genomes option.

2 (1)

Click install.

3 (1)

Navigate to the Installed tab and click on the installed wf-bacterial-genomes workflow.

4 (1)

可选操作

If the workflow was already installed, check for updates by clicking 'Update workflow'.

Ensure you are using v1.3.0 or newer of the workflow. We recommend running the latest version of our workflows for the best results.

Update workflow no miss

Update workflow no miss II

Click on Run this workflow to open the launch wizard.

Run this workflow no miss

Set up your run by uploading your FASTQ file in the Input Options. We recommend keeping the default settings for the other parameter options.

6 (1)

In Sample Options, provide a sample sheet to match sample folder names (e.g. barcode01) to a sample name alias to be used in output reports.

You may specify any number of sample (barcode) folders in your sample sheet.

select-sample-options

To enable the isolates mode, tick the isolates checkbox.

7 (1)

In Advanced Options unselect Prokka annotation.

Optional setting: Set Flye genome size to 5000000, and Flye asm coverage to 50 (these settings are optional but will speed up run time).

select-advanced-options

可选操作

In Miscellaneous Options, set number of CPU threads.

Navigate to the 'Nextflow configuration' tab to assign a 'Run name' to your analysis as an identifier.

Workflow run name no miss

Click Launch workflow.

Ensure all parameter options have green ticks.

7 (2)

Once the workflow finishes, a report will be produced.

可选操作

Test data is available on Github to test the wf-bacterial-genomes workflow.

The test data can be found in the following repository: wf-bacterial-genomes test data

7. 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2-8℃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。

请按照“回收程序”清洗好芯片，以便送回Oxford Nanopore。

您可在 此处找到回收测序芯片的说明。

请注意： 在将测序芯片寄回之前，请使用去离子水对每张芯片进行冲洗。

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。

8. Issues during DNA/RNA extraction and library preparation

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.

Low DNA recovery after AMPure bead clean-up

Observation Possible cause Comments and actions
Low recovery DNA loss due to a lower than intended AMPure beads-to-sample ratio 1. AMPure beads settle quickly, so ensure they are well resuspended before adding them to the sample.

2. When the AMPure beads-to-sample ratio is lower than 0.4:1, DNA fragments of any size will be lost during the clean-up.
Low recovery DNA fragments are shorter than expected The lower the AMPure beads-to-sample ratio, the more stringent the selection against short fragments. Please always determine the input DNA length on an agarose gel (or other gel electrophoresis methods) and then calculate the appropriate amount of AMPure beads to use. SPRI cleanup
Low recovery after end-prep The wash step used ethanol <80% DNA will be eluted from the beads when using ethanol <80%. Make sure to use the correct percentage.

9. Issues during the sequencing run using a Rapid-based sequencing kit

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 10–50 fmol of good quality library can be loaded on to a MinION Mk1B/GridION 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 Rapid PCR Barcoding Kit V14 was used, and sequencing adapters did not attach to the DNA Make sure to closely follow the protocol and use the correct volumes and incubation temperatures. A Lambda control library can be prepared to test the integrity of reagents.
Pore occupancy close to 0 No tether on the flow cell Tethers are added during flow cell priming (FCT tube). Make sure FCT was added to 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。

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 MK1B温度控制的更多信息，请参考此 FAQ （常见问题）文档。

Last updated: 9/30/2024

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