PCR tiling of SARS-CoV-2 virus - automated Agilent (SQK-RBK110.96 with EXP-MRT001)

概览

For Research Use Only

This protocol uses extracted RNA samples in an automated library preparation using the Agilent Bravo to increase reproducibility and reduce human error. Multiple samples can be prepared simultaneously for high sequencing output.

Document version: MRTA_9157_v110_revG_18May2022

1. Overview of the protocol

重要

This protocol is a work in progress and some details are expected to change over time. Please make sure you always use the most recent version of the protocol and scripts.

The PCR tiling of SARS-CoV-2 virus - automated Agilent Bravo (SQK-RBK110.96 with EXP-MRT001) protocol is an automated version of the PCR tiling of SARS-CoV-2 virus with rapid barcoding and Midnight RT PCR Expansion (SQK-RBK110.96 and EXP-MRT001) using the Agilent Bravo liquid handling robot.

Introduction to the protocol

To enable support for the rapidly expanding user requests, the team at Oxford Nanopore Technologies have put together an updated workflow based on the ARTIC Network protocols and analysis methods. The protocol uses Oxford Nanopore Technologies' Rapid Barcoding Kit 96 (SQK-RBK110.96) and Midnight RT PCR Expansion (EXP-MRT001) for barcoding and library preparation.

We have developed this automated protocol on the Agilent Bravo liquid handling robot. The majority of the process is automated with minimal hands-on time which is required for sample quantification and deck re-loading.

While this protocol is available in the Nanopore Community, we kindly ask users to ensure they are citing the members of the ARTIC network who have been behind the development of these methods.

This protocol is similar to the ARTIC amplicon sequencing protocol for MinION for SARS-CoV-2 v3 (LoCost) by Josh Quick and the method used in Freed et al., 2020. The protocol generates amplicons in a tiled fashion across the whole SARS-CoV-2 genome.

To generate tiled PCR amplicons from the SARS-CoV-2 viral cDNA for use with the Rapid Barcoding Kit 96 (SQK-RBK110.96), primers were designed by Freed et al., 2020 using Primal Scheme. These primers are in the Midnight RT PCR Expansion (EXP-MRT001) and are designed to generate 1.2 kb amplicons. Primer sequences can be found here.

Steps in the sequencing workflow:

Prepare for your experiment

you will need to:

Before starting - Manual steps:

  • Extract your RNA
  • Ensure you have your sequencing kit, the correct equipment and reagents
  • Prepare your reagents, samples and labware to load on the Agilent Bravo
  • 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:

Automated steps:

  • Reverse transcribe your RNA samples with random hexamers
  • Amplify the samples by tiled PCR using separate primer pools
  • Combine the primer pools
  • Attach Rapid Barcodes supplied in the kit to the DNA ends, pool the samples and SPRI purify

__Manual steps:__
  • Quantify your DNA library as a quality control
  • Prime the flow cell and load your DNA library into the flow cell

Overview of library preparation workflow:

The image below is representative of the steps that take place in the automated runs for X96 samples.

ARTIC SQK-RBK110.96 96 samples spike-seq

Note: Timings are dependent on number of samples and include hands on time, such as deck loading and sample quantification

Sequencing and analysis

You will need to:

  • Start a sequencing run using the MinKNOW software, selecting SQK-RBK110.96 and EXP-MRT001 in kit selection, which will collect raw data from the device and convert it into basecalled reads

Timings

Note: Timings are approximate and subject to change with updates.

Process X24 samples X48 samples X96 samples Hands-on time
Deck set-up ~30 minutes
Process 1:
cDNA synthesis
8 minutes 16 minutes 24 minutes
Off-Deck thermocyler 17 minutes 17 minutes 17 minutes
Process 2:
cDNA amplification
10 minutes 16 minutes 21 minutes
Off-Deck thermocyler ~3 hours 30 minutes ~3 hours 30 minutes ~3 hours 30 minutes
Process 3:
Rapid Barcoding
5 minutes 8 minutes 10 minutes
Off-Deck thermocyler 8 minutes 8 minutes 8 minutes
Process 4:
cDNA amplicon pooling/cleanup
40 minutes 50 minutes 60 minutes
Quantification ~10 minutes
Total 4 hours 58 minutes 5 hours 25 minutes 5 hours 50 minutes ~40 minutes

Nomenclature for automation protocol

Throughout this document, 'Protocol' is defined as the assay on the whole and 'Run' refers to the individual scripts for the automated liquid handling robot, which are specific to indicated protocol step(s).

Before starting

This protocol outlines how to carry out PCR tiling of SARS-CoV-2 viral RNA samples on a 96-well plate using the Rapid Barcoding Kit 96 (SQK-RBK110.96) with the Midnight RT PCR Expansion (EXP-MRT001) using the Agilent Bravo liquid handling robot.

When processing multiple samples at once, we recommend making master mixes following the indicated volumes to account for the necessary excess. We also recommend using a template-free pre-PCR hood for making up the master mixes, and a separate template pre-PCR hood for handling the samples. It is important to clean and/or UV irradiate these hoods between sample batches. Furthermore, to track and monitor cross-contamination events, it is important to run a negative control reaction at the reverse transcription stage using nuclease-free water instead of sample, and carrying this control through the rest of the prep.

All post-PCR procedures should be carried out in a separate area to the pre-PCR preparation, with dedicated equipment for liquid handling in each area to minimise risk of contamination.

If only one liquid handling robot is available, both pre-PCR and post-PCR sections of the assay can be performed in the same robot. In this scenario, cleaning the equipment thoroughly between runs is required and we recommend validating the process for your unique set-up.

重要

Compatibility of this protocol

This protocol should only be used in combination with:

  • Rapid Barcoding Kit 96 (SQK-RBK110.96)
  • Midnight RT PCR Expansion (EXP-MRT001)
  • R9.4.1 flow cells (FLO-MIN106)
  • Flow Cell Wash Kit (EXP-WSH004)

2. Equipment and consumables

材料
  • Input RNA in 10 mM Tris-HCl, pH 8.0
  • Rapid Barcoding Kit 96 (SQK-RBK110.96)
  • Midnight RT PCR Expansion (EXP-MRT001)

耗材
  • Nuclease-free water (e.g. 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
  • 2 ml Eppendorf DNA LoBind 离心管
  • 5 ml Eppendorf DNA LoBind tubes
  • Bravo Lab Disposable Pipette Tips 250 µl - compatible with Bravo 96LT head (19477-022)
  • Arvensis B-Frame BIOCOMPOSITE 96 Well PCR Plate Fully Skirted Low Profile 0.2 ml wells
  • Hard-Shell® 96-Well PCR Plates, low profile, thin-walled, skirted, white/clear (Bio-Rad, Cat # HSP9601)
  • 96-well 0.8 ml MIDI plate (we recommend Abgene™ 96 Well 0.8 ml Polypropylene Deepwell Storage Plate: ThermoFisher, Cat # AB0859)
  • PCR plate seals

仪器
  • Agilent Bravo liquid handling robot
  • Centrifuge capable of taking 96-well plates
  • 迷你离心机
  • 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
  • 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
  • Eppendorf 5424 离心机(或等效器材)
  • PCR hood with UV steriliser (optional but recommended to reduce cross-contamination)
  • PCR-Cooler (Eppendorf)
  • Stepper pipette and tips

Rapid Barcoding Kit 96 (SQK-RBK110.96) contents

RBK110.96 kit contents

Name Acronym Cap colour No. of vials Fill volume per vial (µl)
Rapid Barcode plate RB96 - 3 plates 8 µl per well
AMPure XP Beads AXP Brown 3 1,200
Sequencing Buffer II SBII Red 1 500
Rapid Adapter F RAP-F Green 1 25
Elution Buffer EB Black 1 500
Loading Beads II LBII Pink 1 360
Loading Solution LS White cap, pink label 1 400
Flush Tether FLT Purple 1 400
Flush Buffer FB White 1 bottle 15,500

This product contains AMPure XP reagent manufactured by Beckman Coulter, Inc.

Midnight RT PCR Expansion (EXP-MRT001) contents

EXP-MRT001 1

Name Acronym Cap colour Number of vials Fill volume per vial (µl)
LunaScript RT SuperMix LS RT Blue 3 500
Q5 HS Master Mix Q5 Orange 6 1,500
Midnight Primer Pool A MP A White 3 15
Midnight Primer Pool B MP B Clear 3 15

Midnight Primer sequences

As mutations in SARS-CoV-2 variants emerge amplicon drop out may be observed; for users wishing to design their own primer spike-ins to address this we suggest adding to the appropriate primer pool at a final concentration between 3.33 µM and 6.66 µM.

Below are the sequences for the V3 primer scheme used in the Midnight RT PCR Expansion.

Pool A

Primer name Primer Sequence
SARSCoV_1200_1_LEFT ACCAACCAACTTTCGATCTCTTGT
SARSCoV_1200_1_RIGHT GGTTGCATTCATTTGGTGACGC
SARSCoV_1200_3_LEFT GGCTTGAAGAGAAGTTTAAGGAAGGT
SARSCoV_1200_3_RIGHT GATTGTCCTCACTGCCGTCTTG
SARSCoV_1200_5_LEFT ACCTACTAAAAAGGCTGGTGGC
SARSCoV_1200_5_RIGHT AGCATCTTGTAGAGCAGGTGGA
SARSCoV_1200_7_LEFT ACCTGGTGTATACGTTGTCTTTGG
SARSCoV_1200_7_RIGHT GCTGAAATCGGGGCCATTTGTA
SARSCoV_1200_9_LEFT AGAAGTTACTGGCGATAGTTGTAATAACT
SARSCoV_1200_9_RIGHT TGCTGATATGTCCAAAGCACCA
SARSCoV_1200_11_LEFT AGACACCTAAGTATAAGTTTGTTCGCA
SARSCoV_1200_11_RIGHT GCCCACATGGAAATGGCTTGAT
SARSCoV_1200_13_LEFT ACCTCTTACAACAGCAGCCAAAC
SARSCoV_1200_13_RIGHT CGTCCTTTTCTTGGAAGCGACA
SARSCoV_1200_15_LEFT TTTTAAGGAATTACTTGTGTATGCTGCT
SARSCoV_1200_15_RIGHT ACACACAACAGCATCGTCAGAG
SARSCoV_1200_17_LEFT TCAAGCTTTTTGCAGCAGAAACG
SARSCoV_1200_17_RIGHT CCAAGCAGGGTTACGTGTAAGG
SARSCoV_1200_19_LEFT GGCACATGGCTTTGAGTTGACA
SARSCoV_1200_19_RIGHT CCTGTTGTCCATCAAAGTGTCCC
SARSCoV_1200_21_LEFT TCTGTAGTTTCTAAGGTTGTCAAAGTGA
SARSCoV_1200_21_RIGHT GCAGGGGGTAATTGAGTTCTGG
21_right_spike GTGTATGATTGAGTTCTGGTTGTAAG
SARSCoV_1200_23_LEFT ACTTTAGAGTCCAACCAACAGAATCT
23_left_spike ACTTTAGAGTTCAACCAACAGAATCT
SARSCoV_1200_23_RIGHT TGACTAGCTACACTACGTGCCC
SARSCoV_1200_25_LEFT TGCTGCTACTAAAATGTCAGAGTGT
SARSCoV_1200_25_RIGHT CATTTCCAGCAAAGCCAAAGCC
SARSCoV_1200_27_LEFT TGGATCACCGGTGGAATTGCTA
SARSCoV_1200_27_RIGHT TGTTCGTTTAGGCGTGACAAGT
SARSCoV_1200_29_LEFT TGAGGGAGCCTTGAATACACCA
SARSCoV_1200_29_RIGHT TAGGCAGCTCTCCCTAGCATTG

Pool B

Primer name Primer sequences
SARSCoV_1200_2_LEFT CCATAATCAAGACTATTCAACCAAGGGT
SARSCoV_1200_2_RIGHT ACAGGTGACAATTTGTCCACCG
SARSCoV_1200_4_LEFT GGAATTTGGTGCCACTTCTGCT
SARSCoV_1200_4_RIGHT CCTGACCCGGGTAAGTGGTTAT
SARSCoV_1200_6_LEFT ACTTCTATTAAATGGGCAGATAACAACTG
SARSCoV_1200_6_RIGHT GATTATCCATTCCCTGCGCGTC
SARSCoV_1200_8_LEFT CAATCATGCAATTGTTTTTCAGCTATTTTG
SARSCoV_1200_8_RIGHT TGACTTTTTGCTACCTGCGCAT
SARSCoV_1200_10_LEFT TTTACCAGGAGTTTTCTGTGGTGT
SARSCoV_1200_10_RIGHT TGGGCCTCATAGCACATTGGTA
SARSCoV_1200_12_LEFT ATGGTGCTAGGAGAGTGTGGAC
SARSCoV_1200_12_RIGHT GGATTTCCCACAATGCTGATGC
SARSCoV_1200_14_LEFT ACAGGCACTAGTACTGATGTCGT
SARSCoV_1200_14_RIGHT GTGCAGCTACTGAAAAGCACGT
SARSCoV_1200_16_LEFT ACAACACAGACTTTATGAGTGTCTCT
SARSCoV_1200_16_RIGHT CTCTGTCAGACAGCACTTCACG
SARSCoV_1200_18_LEFT GCACATAAAGACAAATCAGCTCAATGC
SARSCoV_1200_18_RIGHT TGTCTGAAGCAGTGGAAAAGCA
SARSCoV_1200_20_LEFT ACAATTTGATACTTATAACCTCTGGAACAC
SARSCoV_1200_20_RIGHT GATTAGGCATAGCAACACCCGG
SARSCoV_1200_22_LEFT GTGATGTTCTTGTTAACAACTAAACGAACA
SARSCoV_1200_22_RIGHT AACAGATGCAAATCTGGTGGCG
22_right_spike AACAGATGCAAATTTGGTGGCG
SARSCoV_1200_24_LEFT GCTGAACATGTCAACAACTCATATGA
24_left_spike GCTGAATATGTCAACAACTCATATGA
SARSCoV_1200_24_RIGHT ATGAGGTGCTGACTGAGGGAAG
SARSCoV_1200_26_LEFT GCCTTGAAGCCCCTTTTCTCTA
SARSCoV_1200_26_RIGHT AATGACCACATGGAACGCGTAC
SARSCoV_1200_28_LEFT TTTGTGCTTTTTAGCCTTTCTGCT
SARSCoV_1200_28_RIGHT GTTTGGCCTTGTTGTTGTTGGC
SARSCoV_1200_28_LEFT_27837T TTTGTGCTTTTTAGCCTTTCTGTT

Rapid barcode sequences

Component Sequence
RB01 AAGAAAGTTGTCGGTGTCTTTGTG
RB02 TCGATTCCGTTTGTAGTCGTCTGT
RB03 GAGTCTTGTGTCCCAGTTACCAGG
RB04 TTCGGATTCTATCGTGTTTCCCTA
RB05 CTTGTCCAGGGTTTGTGTAACCTT
RB06 TTCTCGCAAAGGCAGAAAGTAGTC
RB07 GTGTTACCGTGGGAATGAATCCTT
RB08 TTCAGGGAACAAACCAAGTTACGT
RB09 AACTAGGCACAGCGAGTCTTGGTT
RB10 AAGCGTTGAAACCTTTGTCCTCTC
RB11 GTTTCATCTATCGGAGGGAATGGA
RB12 CAGGTAGAAAGAAGCAGAATCGGA
RB13 AGAACGACTTCCATACTCGTGTGA
RB14 AACGAGTCTCTTGGGACCCATAGA
RB15 AGGTCTACCTCGCTAACACCACTG
RB16 CGTCAACTGACAGTGGTTCGTACT
RB17 ACCCTCCAGGAAAGTACCTCTGAT
RB18 CCAAACCCAACAACCTAGATAGGC
RB19 GTTCCTCGTGCAGTGTCAAGAGAT
RB20 TTGCGTCCTGTTACGAGAACTCAT
RB21 GAGCCTCTCATTGTCCGTTCTCTA
RB22 ACCACTGCCATGTATCAAAGTACG
RB23 CTTACTACCCAGTGAACCTCCTCG
RB24 GCATAGTTCTGCATGATGGGTTAG
RB25 GTAAGTTGGGTATGCAACGCAATG
RB26 CATACAGCGACTACGCATTCTCAT
RB27 CGACGGTTAGATTCACCTCTTACA
RB28 TGAAACCTAAGAAGGCACCGTATC
RB29 CTAGACACCTTGGGTTGACAGACC
RB30 TCAGTGAGGATCTACTTCGACCCA
RB31 TGCGTACAGCAATCAGTTACATTG
RB32 CCAGTAGAAGTCCGACAACGTCAT
RB33 CAGACTTGGTACGGTTGGGTAACT
RB34 GGACGAAGAACTCAAGTCAAAGGC
RB35 CTACTTACGAAGCTGAGGGACTGC
RB36 ATGTCCCAGTTAGAGGAGGAAACA
RB37 GCTTGCGATTGATGCTTAGTATCA
RB38 ACCACAGGAGGACGATACAGAGAA
RB39 CCACAGTGTCAACTAGAGCCTCTC
RB40 TAGTTTGGATGACCAAGGATAGCC
RB41 GGAGTTCGTCCAGAGAAGTACACG
RB42 CTACGTGTAAGGCATACCTGCCAG
RB43 CTTTCGTTGTTGACTCGACGGTAG
RB44 AGTAGAAAGGGTTCCTTCCCACTC
RB45 GATCCAACAGAGATGCCTTCAGTG
RB46 GCTGTGTTCCACTTCATTCTCCTG
RB47 GTGCAACTTTCCCACAGGTAGTTC
RB48 CATCTGGAACGTGGTACACCTGTA
RB49 ACTGGTGCAGCTTTGAACATCTAG
RB50 ATGGACTTTGGTAACTTCCTGCGT
RB51 GTTGAATGAGCCTACTGGGTCCTC
RB52 TGAGAGACAAGATTGTTCGTGGAC
RB53 AGATTCAGACCGTCTCATGCAAAG
RB54 CAAGAGCTTTGACTAAGGAGCATG
RB55 TGGAAGATGAGACCCTGATCTACG
RB56 TCACTACTCAACAGGTGGCATGAA
RB57 GCTAGGTCAATCTCCTTCGGAAGT
RB58 CAGGTTACTCCTCCGTGAGTCTGA
RB59 TCAATCAAGAAGGGAAAGCAAGGT
RB60 CATGTTCAACCAAGGCTTCTATGG
RB61 AGAGGGTACTATGTGCCTCAGCAC
RB62 CACCCACACTTACTTCAGGACGTA
RB63 TTCTGAAGTTCCTGGGTCTTGAAC
RB64 GACAGACACCGTTCATCGACTTTC
RB65 TTCTCAGTCTTCCTCCAGACAAGG
RB66 CCGATCCTTGTGGCTTCTAACTTC
RB67 GTTTGTCATACTCGTGTGCTCACC
RB68 GAATCTAAGCAAACACGAAGGTGG
RB69 TACAGTCCGAGCCTCATGTGATCT
RB70 ACCGAGATCCTACGAATGGAGTGT
RB71 CCTGGGAGCATCAGGTAGTAACAG
RB72 TAGCTGACTGTCTTCCATACCGAC
RB73 AAGAAACAGGATGACAGAACCCTC
RB74 TACAAGCATCCCAACACTTCCACT
RB75 GACCATTGTGATGAACCCTGTTGT
RB76 ATGCTTGTTACATCAACCCTGGAC
RB77 CGACCTGTTTCTCAGGGATACAAC
RB78 AACAACCGAACCTTTGAATCAGAA
RB79 TCTCGGAGATAGTTCTCACTGCTG
RB80 CGGATGAACATAGGATAGCGATTC
RB81 CCTCATCTTGTGAAGTTGTTTCGG
RB82 ACGGTATGTCGAGTTCCAGGACTA
RB83 TGGCTTGATCTAGGTAAGGTCGAA
RB84 GTAGTGGACCTAGAACCTGTGCCA
RB85 AACGGAGGAGTTAGTTGGATGATC
RB86 AGGTGATCCCAACAAGCGTAAGTA
RB87 TACATGCTCCTGTTGTTAGGGAGG
RB88 TCTTCTACTACCGATCCGAAGCAG
RB89 ACAGCATCAATGTTTGGCTAGTTG
RB90 GATGTAGAGGGTACGGTTTGAGGC
RB91 GGCTCCATAGGAACTCACGCTACT
RB92 TTGTGAGTGGAAAGATACAGGACC
RB93 AGTTTCCATCACTTCAGACTTGGG
RB94 GATTGTCCTCAAACTGCCACCTAC
RB95 CCTGTCTGGAAGAAGAATGGACTT
RB96 CTGAACGGTCATAGAGTCCACCAT

3. 计算机要求及软件 (2)

MinION Mk1B的IT配置要求

请为MinION Mk1B配备一台高规格的计算机或笔记本电脑,以适配数据采集的速度。您可以在MinION Mk1B的IT配置要求文件中了解更多。

MinION Mk1C的IT配置要求

MinION Mk1C是一款集计算功能和触控屏幕于一体的便携式测序分析仪,它无需依赖任何额外设备,即可生成并分析纳米孔测序数据。您可以在 MinION Mk1C的IT配置要求文件中了解更多。

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 the EPI2ME Platform protocol.

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. Pre-PCR

材料
  • Input RNA in 10 mM Tris-HCl, pH 8.0
  • Midnight Primer Pool A (MP A)
  • Midnight Primer Pool B (MP B)

耗材
  • Arvensis B-Frame BIOCOMPOSITE 96 Well PCR Plate Fully Skirted Low Profile 0.2 ml wells
  • Bravo Lab Disposable Pipette Tips 250 µl - compatible with Bravo 96LT head (19477-022)
  • PCR plate seals
  • Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
  • LunaScript RT SuperMix (LS RT)
  • 1.5 ml Eppendorf DNA LoBind 离心管
  • Q5 HS Master Mix (Q5)

仪器
  • Ice bucket with ice
  • PCR hood with UV steriliser (optional but recommended to reduce cross-contamination)
  • Agilent Bravo liquid handling robot
  • P1000 移液枪和枪头
  • P200 移液枪和枪头
  • P20 移液枪和枪头
  • P2 移液枪和枪头
  • 迷你离心机
  • 热循环仪
  • Centrifuge capable of taking 96-well plates

Run setup:

Turn on the Agilent Thermocube and set Position 4 to 4°C.

To cool position 4 prior to the run: open the file, under the deck layout, and tick the box near the block you want to cool. Set up the temperature and hit 'Run' to execute.

Agilent Themocube CPAC Temperature

Thaw and keep the samples, LunaScript, Q5 Hot Start Master mix, Midnight Primer Pool A (MPA) and Midnight Primer Pool B (MPB) on ice.

重要

To minimise risk of contamination, we recommend handling the Lunascript and primers in a clean template-free PCR hood.

重要

Prior to use, ensure all reagents have been thoroughly mixed by performing 20-30 full-volume pipette mixes.

Note: This is especially important with the Q5 Hot start master mix as the contents can precipitate following freeze-thaw cylces.

Take care when mixing and vortexing as this will introduce air bubbles, resulting in loss of volume.

In the template-free pre-PCR hood, prepare the following Primer master mixes in 1.5 ml Eppendorf DNA LoBind tubes and mix thoroughly as follows:

For x24 samples:

| Reagent | Pool A | Pool B | | --- | --- | --- | --- | --- | | Nuclease-free water | 172 µl | 172 µl | | Midnight Primer Pool A (MP A) | 2 µl | - | | Midnight Primer Pool B (MP B) | - | 2 µl | | Q5 HS Master Mix (Q5) | 102 µl | 102 µl | | Total | 276 µl | 276 µl |

For x48 samples:

| Reagent | Pool A | Pool B | | --- | --- | --- | --- | --- | | Nuclease-free water | 344 µl | 344 µl | | Midnight Primer Pool A (MP A) | 3 µl | - | | Midnight Primer Pool B (MP B) | - | 3 µl | | Q5 HS Master Mix (Q5) | 203 µl | 203 µl | | Total | 550 µl | 550 µl |

For x96 samples:

| Reagent | Pool A | Pool B | | --- | --- | --- | --- | --- | | Nuclease-free water | 687 µl | 687 µl | | Midnight Primer Pool A (MP A) | 6 µl | - | | Midnight Primer Pool B (MP B) | - | 6 µl | | Q5 HS Master Mix (Q5) | 407 µl | 407 µl | | Total | 1,100 µl | 1,100 µl |

Note: Taking care not to introduce air bubbles, pipette mix 10-15 times between each addition and perform a final full-volume pipette mix 10 times.

Keep on ice until use.

In the template-free pre-PCR hood and using a clean Arvensis plate, prepare the reagent input plate as follows:

For x24 samples:

Reagents Agilent PRE x24

For x48 samples:

Reagents Agilent PRE x48

For x96 samples:

Reagents Agilent PRE x96

Note: Take care to not introduce air bubbles while aliquoting the reagents into the Arvensis plate.

Once complete seal the plate and keep on ice until ready to transfer over to the Agilent Bravo robot.

In a pre-PCR hood and using a clean Arvensis plate, prepare the RNA sample input plate as follows:

For x24 samples:

Sample input x24 MRT

For x48 samples:

Sample input x48 MRT

For x96 samples:

Sample input x96 MRT

Note: Take care to not introduce air bubbles while aliquoting the samples into the Arvensis plate.

Once complete seal the plate and keep on ice until ready to transfer over to the Agilent Bravo robot.

On the Agilent Bravo, select the 'cDNA and Multiplex (X) samples' protocol, where (X) indicates the number of samples to be processed.

Set the number of columns of samples and the PCR plate type.

  • For X24 samples, select 3
  • For X48 samples, select 6
  • For X96 samples, select 12

Select 'Display Deck Layout'.

Add the labware, sample plate and reagent input plate as indicated on the form display.

For X24 samples:

Agilent pre deck layout x24

For X48 samples:

Agilent pre deck layout x48

For X96 samples:

Agilent pre deck layout x96

Select 'Run Protocol'.

To start the run, select 'Ok' from the figure below:

Agilent Bravo start run OK

After the Agilent Bravo has added the 2 µl of LunaScript to the samples, the robot will stop with the the following message:

Agilent Bravo RT PCR Wide

Remove the sample plate containing the LunaScript from the Agilent Bravo, seal it and spin it down.

Place the sample plate in a Thermal cycler and incubate using the following program:

Step Temperature Time Cycles
Primer annealing 25°C 2 min 1
cDNA synthesis 55°C 10 min 1
Heat inactivation 95°C 1 min 1
Hold 4°C

After placing the sample plate in the Thermal cycler, press "Continue" on the Agilent Bravo to start the addition of the Primer master mixes into a clean Arvensis plate.

Once the thermal cycler has completed the cDNA synthesis, remove the sample plate and spin down.

When prompted, place the sample plate back in the Agilent Bravo deck into position 4.

After the Agilent Bravo has added the 2.5 µl of RT to the Primer master mix plate/plates, the robot will stop with the the following message:

Agilent PCR AMP WIDE

Remove the PCR plate (or plates if processing X96 samples) from the Agilent Bravo, seal it and spin it down.

Place in the thermal cycler and incubate using the following program, with the heated lid set to 105°C:

Step Temperature Time Cycles
Initial denaturation 98°C 30 sec 1
Denaturation

Annealing and extension
98°C

61°C
65°C
15 sec

2 min
3 min

35
Hold 4°C
步骤结束

Once the PCR amplification is complete, remove the plate(s) from the thermal cycler and spin down. The plate(s) will be taken forward to the Post-PCR section of the protocol.

If necessary, the protocol can be paused at this point. The samples should be kept at 4°C and can be stored overnight.

5. Post-PCR

材料
  • Rapid Barcode Plate (RB96)
  • AMPure XP Beads (AXP, or SPRI)
  • Elution Buffer from the Oxford Nanopore kit (EB)
  • Rapid Adapter F (RAP F)

耗材
  • Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
  • 新制备的80%乙醇(用无核酸酶水配制)
  • Bravo Lab Disposable Pipette Tips 250 µl - compatible with Bravo 96LT head (19477-022)
  • Arvensis B-Frame BIOCOMPOSITE 96 Well PCR Plate Fully Skirted Low Profile 0.2 ml wells
  • 96-well 0.8 ml MIDI plate (we recommend Abgene™ 96 Well 0.8 ml Polypropylene Deepwell Storage Plate: ThermoFisher, Cat # AB0859)
  • PCR plate seals
  • Qubit™ 分析管(Invitrogen, Q32856)
  • Qubit dsDNA HS Assay(双链DNA高灵敏度检测)试剂盒(Invitrogen, Q32851)

仪器
  • Agilent Bravo liquid handling robot
  • 热循环仪
  • Centrifuge capable of taking 96-well plates
  • P1000移液枪和枪头
  • P200 移液枪和枪头
  • P20 pipette and tips
  • P2 移液枪和枪头
  • Qubit荧光计(或用于质控检测的等效仪器)

Rapid Barcoding:

Select the 'Barcoding and clean-up' program on the Agilent Bravo.

Set the number of columns for your run:

  • For three columns of each primer pool (6 in total), select 3 columns.
  • For six columns of each primer pool (12 in total), select 6 columns.
  • For twelve columns of each primer pool (24 in total), select 12 columns.

Select 'Display Deck Layout'.

Add the Primer plates, the Rapid barcode plate and the labware as indicated on the display.

Select 'Run protocol'.

To start the run, select 'Ok' from the figure below:

Agilent Bravo start run OK

可选操作

For runs using X24 or X48 samples, you will need to enter the starting index plate column for the RBK plate when prompted.

For example: If your barcode starts at column 4, enter "4" as seen in the figure below.

Set initial value variables WIDE

After the Agilent Bravo has completed the Rapid Barcoding plate, the robot will stop with the following message:

Agilent Bravo POST RBK Wide

Remove the barcoded sample plate from the Agilent Bravo, seal it and spin it down.

Place the barcoded sample plate in the thermal cycler and incubate at 30°C for 2 minutes and then at 80°C for 2 minutes.

Sample pooling and clean-up:

Follow the instructions for the Agilent Bravo deck set-up on the screen prompt.

Agilent Bravo POST RBK Wide

Resuspend the AMPure XP beads by vortexing.

While the barcoding plate is in the thermal cycler, using a clean MIDI 96 deep-well plate prepare the reagents as follows:

For X24 samples:

Reagents Agilent POST x24

For X48 samples:

Reagents Agilent POST x48

For X96 samples:

Reagents Agilent POST x96

Note: Take care to not introduce air bubbles while aliquoting the reagents into the deep-well plate.

Ensure all reagents are properly mixed prior to use.

When preparing the plates, well A12 will be empty. This is where the final elution will be found at the end of the Agilent Bravo run.

Prepare the deck of the Agilent Bravo with the reagent plate and labware as follows:

For X24 or X48 samples:

Agilent POST deck layout x24 48

For X96 samples:

Agilent POST deck layout x96

Once the thermal cycler has finished, remove the plate and spin it down.

Remove the seal on the rapid barcoding plate and return it to the allocated position on the Agilent Bravo deck.

Select "Continue" on the Agilent Bravo screen prompt to continue the run.

Ensure all of the labware and reagent plates are in the correct positions, you have removed all of the lids and the plates are unsealed before continuing.

After the run ends, the final library can be collected from position A12 in the deep-well plate. Remove and retain the final elution into a clean 1.5 ml Eppendorf DNA LoBind tube

Quantify DNA concentration of the final elution by using the Qubit dsDNA HS Assay Kit.

步骤结束

The quantified library is used for loading into the MinION flow cell. Store the library on ice until ready to load.

6. Priming and loading the SpotON Flow Cell

材料
  • Flush Buffer (FB)
  • Flush Tether (FLT)
  • Loading Beads II (LBII)
  • Sequencing Buffer II (SBII)
  • Loading Solution (LS)

耗材
  • 1.5 ml Eppendorf DNA LoBind 离心管

仪器
  • MinION device
  • SpotON Flow Cell
  • 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
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

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) or Loading Solution (LS, if using), Flush Tether (FLT) and Flush Buffer (FB) at room temperature before mixing the reagents by vortexing, and spin down the SBII and FLT at room temperature.

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

Open the MinION device lid and slide the flow cell under the clip.

Press down firmly on the flow cell to ensure correct thermal and electrical contact.

Flow Cell Loading Diagrams Step 1a

Flow Cell Loading Diagrams Step 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。

Slide the priming port cover clockwise to open the priming port.

Flow Cell Loading Diagrams Step 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

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) 37.5 µl
Loading Beads II (LBII) mixed immediately before use, or Loading Solution (LS), if using 25.5 µl
DNA library 12 µl
Total 75 µl

Note: Load the library onto the flow cell immediately after adding the Sequencing Buffer II (SBII) and Loading Beads II (LBII) because the fuel in the buffer will start to be consumed by the adapter.

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 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。

7. 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。

重要

Required settings in MinKNOW

The correct barcoding parameters must be set up on MinKNOW prior to the sequencing run. During the run setup, in the Analysis tab:

  1. Enable Barcoding.
  2. Select Edit options.
  3. Enable Mid-read barcode filtering.
  4. Enable Override minimum barcoding score and set the value to 60.
  5. Enable Override minimum mid-read barcoding score and set the value to 50.

MRT Run setup analysis - Barcoding highlights

MRT Run setup Barcoding options

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: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 实验指南本地分析 部分操作。

8. Downstream analysis

Recommended pipeline analysis

The wf-artic is a bioinformatics workflow for the analysis of ARTIC sequencing data prepared using the Midnight protocol. The bioinformatics workflow is orchestrated by the Nextflow software. Nextflow is a publicly available and open-source project that enables the execution of scientific workflows in a scalable and reproducible way. The software is natively supported on the GridION device and can be simply installed on most Linux computers and servers. The installation is outlined later in the document.

The Midnight analysis uses the ARTIC bioinformatics workflow.

Demultiplexed sequence reads are processed using the ARTIC FieldBioinformatics software that has been subtly modified for the analysis of FASTQ sequences prepared using Oxford Nanopore rapid sequencing kits. The other modification to the ARTIC workflow is the use of a primer scheme that defines the sequencing primers used by the Midnight protocol and their genomic locations on the SARS-CoV-2 genome.

The wf-artic workflow includes other analytical steps that include cladistic analysis using Nextclade and strain assignment using Pangolin. The data facets included in the report are parameterised and additional information such as plots of depth-of-coverage across the reference genome is optional.

The complete source for wf-artic is linked and the Nextflow software will download the scripts and logic flow from this location.

On GridION devices, the wf-artic workflow will start automatically after sequencing. However, on other devices, this will have to be started manually as outlined further on this page under 'Running a Midnight analysis'.

Software set up and installation

The wf-artic workflow requires the Nextflow and Docker software to have been installed. The EPI2ME quickstart guide provides instructions for the installation of these requirements for GridION, PromethION and general Ubuntu Linux users and provides a little more introduction to the Nextflow software.

Automatic start on GridION:

To set up the Midnight analysis to start automatically after sequencing on GridION, select the Rapid Barcoding Kit 96 (SQK-RBK110.96) kit with the Midnight RT PCR Expansion (EXP-MRT001) pack on MinKNOW when setting up a sequencing run.

When the workflow has finished, the relevant analysis files will be available in the following output folder:

processing/artic/artic_DATE_TIME_67195e17

Post-run analysis on GridION:

The Midnight analysis can also be started post-run on GridION:

  1. On the start page, click 'Analysis'
  2. Click 'Workflow'
  3. From the dropdown menu, select 'post_processing/artic/artic'
  4. Select your input folder with the sequencing data and the location for the output folder

Midnight gridion workflow

Using Linux command line:

The wf-artic workflow can be run from the Linux command line. The workflow can be installed or updated with the command:

$ nextflow pull epi2me-labs/wf-artic

Demultiplexing of multiple barcoded samples

The wf-artic requires FASTQ format sequence data that has already been demultiplexed. Sequences can either be demultiplexed directly in the MinKNOW software or as a post-sequencing step by the guppy_barcoder software provided by the Guppy software.

The Midnight protocol uses a rapid barcoding kit; it is therefore important to note that the demultiplexing step must not require barcodes at both ends of the sequence.

The expected input for wf-artic is a folder of folders as shown below. Each of the barcode folders should contain the FASTQ sequence data and files may either be uncompressed or gzipped.

$ tree -d MidnightFastq/

MidnightFastq/

├── barcode01

├── barcode02

├── barcode03

├── barcode04

├── barcode05

├── barcode06

└── unclassified

Running a Midnight analysis

The reference command for running a Midnight analysis is as follows. The parameters are explained further on in the document.

nextflow run epi2me-labs/wf-artic \

--scheme_name SARS-CoV-2 \

--scheme_version V1200 \

--min_len 200 \

--max_len 1100 \

--out_dir PATH_TO_OUTPUT \

--fastq PATH_TO_FASTQ_PASS \

-work-dir PATH_TO_INTERMEDIATE_FILES

Type the command into you linux terminal and press enter.

Picture1Midnight

Nextflow will describe the analysis as it progresses; the figure above shows an example run from a 48-plex analysis. We can see which processes have completed and the processes that are still running and or queued.

Parameter definitions

  • nextflow run epi2me-labs/wf-artic An instruction to use the Nextflow software to run a workflow, which is further explained here.

  • --scheme_name SARS-CoV-2 An instruction for the ARTIC software to use the primer scheme that corresponds to the amplicons tiled across the whole SARS-CoV-2 genome.

  • --scheme_version V1200 This defines the version of the ARTIC primers to use. The Midnight protocol uses the primer set refered to as V1200.

  • –-min_len 200 This sets the minimum allowed sequence length as 200 nucleotides.

  • –-max_len 1100 This sets the maximum allowed sequence length as 1100 nucleotides.

  • --out_dir PATH_TO_OUTPUT This instructs the Nextflow software where the results should be stored; please change PATH_TO_OUTPUT to the location on your computer where files should be stored.

  • --fastq PATH_TO_FASTQ_PASS This instructs Nextflow which sequences should be used in the analysis. Please change PATH_TO_FASTQ_PASS to an existing fastq_pass folder from a Midnight run.

  • -work_dir PATH TO WORK DIRECTORY Please note the single hyphen; this is a Nextflow parameter. This defines where the intermediate files are stored. This folder may contain a significant amount of information; please see the section on housekeeping.

Other command line parameters

Other commands and options can be provided to the Nextflow command:

  • --samples This describes a sample file that links barcode identifier with sample names. These sample names will be reported in the HTML format report and in the CSV file of genotypes. The sample file should be a comma-delimited file and must contain the column names barcode and sample_name.

  • --help This will display the help-file which describes the available parameters and other information on default values and their meanings.

  • --medaka_model This defines the model that should be used by the Medaka software for variant calling (and thus consensus preparation).

重要

Basecalling model

If you are basecalling using a FAST model, then this should be changed to reflect the appropriate model and version of Guppy used.

  • Default model used: r941_min_hac_variant_g507.
  • If you have used FAST basecalling, please use: r941_min_fast_variant_g507.
  • If HAC basecalling was performed using an earlier version of MinKNOW, please use: r941_min_high_g360.

Result files

Results will be written to the location specified by the --out_dir parameter. These output results include:

  • all_consensus.fasta A multi-FASTA format sequence file containing the consensus sequence for each of the samples investigated. This consensus sequence has been prepared for the whole SARS-CoV-2 genome, not just the spike protein region. The consensus sequence masks the non-spike regions and regions of low sequence coverage with N residues.

  • all_variants.vcf.gz A gzipped VCF file that describes all high-quality genetic variants called by medaka from the sequenced samples.

  • all_variants.vcf.gz.tbi An index file for the gzipped VCF file.

  • consensus_status.txt A tab delimited file that reports whether a consensus sequence has been successfully prepared for a sample, or not.

  • wf-artic-report.html A report summarising these data. This HTML format report also includes the output of the Nextclade software that can be used for a visual inspection of, for example, primer drop out or other qualitative consensus sequence aspects.

Other files are included in the work-directory. This includes per sample VCF files of all genetic variants prior to filtering and other sequences.

Housekeeping and disk usage

The nextflow parameter, -work-dir, was introduced as a parameter to define where the workflow intermediate files are stored. This folder will accumulate a significant number of files that correspond to raw BAM files and other larger intermediates. We recommend this folder to be routinely cleared.

Updating the wf-artic software

Updated versions of the wf-artic software may be released and an alert to the availability of newer workflow versions will be noted by the Nextflow software at run-time.

To update the software:

nextflow pull epi2me-labs/wf-artic

It may be necessary to first delete the cached workflow files. This can be achieved with the command:

nextflow drop -f epi2me-labs/wf-artic

9. 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。

10. 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。

11. 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 MK1B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: 6/29/2023

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