Rapid sequencing gDNA - whole genome amplification (SQK-RAD004)

Version for device: MinION

1. Important This is a Legacy product

   This kit has now been discontinued and we recommend all customers upgrade to the latest chemistry for their relevant kit which is available on the Store. For further information on please see the product update page.

2. Overview

   Oxford Nanopore Technologies' range of sequencing kits is designed to prepare DNA and RNA libraries using 10 ng input material or more. However, users with less than 1 ng can use whole genome amplification to generate more starting material in order to use our sequencing kits. We have previously released a Premium whole genome amplification protocol. This protocol recommends a lengthy amplification step using the REPLI-g Midi Kit (Qiagen, cat # 150043), followed by a T7 endonuclease I digestion and library preparation using the Ligation Sequencing Kit; typically the whole process takes more than a day. We have now developed a much shorter protocol allowing users to go from “cells to sequence” in under two hours. Using the QIAGEN REPLI-g UltraFast Mini Kit, whole genome amplification is performed in ~100 minutes using the REPLI-g UltraFast Mini Kit (Qiagen, cat # 150033) followed by library preparation using the Rapid Sequencing Kit.

   The Rapid method has been designed to generate sequencing libraries from a low number of cells, with minimal sample manipulation and minimal lab equipment, in under 2 hours. However, there is a significant compromise to throughput using this method over the ligation-based library prep, and is generally recommended only for applications where throughput is of low importance.

   

   Although cells can be used in the REPLI-g kit, the chemical lysis step may not be sufficient to release DNA from all cell types. Additional steps to ensure all cells are lysed may be advised, depending on sample type.

3. Input recommendation

   The REPLI-g UltraFast Mini Kit suggests an input of either 300 cells‡, or 1-10 ng‡‡ of gDNA. The method describing how to perform whole genome amplification differs, depending on sample type (cells or DNA), and details for each are provided. Please also refer to manufacturer's guides for further details.

   Notes:
   ‡ Although cells can be used in the REPLI-g kit, the chemical lysis step may not be sufficient to release the DNA from all cell types. Additional steps to ensure all cells are lysed may be required, depending on sample type.
   ‡‡ We have found that it is possible to reduce the input amount to, for example, 10 pg of gDNA. However, lowering the input may require the time of the amplification step to be increased. We recommend amplifying until the DNA concentration is >80 ng/μl (quantification performed using the Qubit dsDNA BR Assay Kit).

4. Introduction to the Rapid whole genome amplification protocol

   This protocol describes how to carry out whole genome amplification and sequencing of genomic DNA using the Rapid Sequencing Kit (SQK-RAD004). It is highly recommended that a Lambda control experiment is completed first to become familiar with the technology.

   Steps in the sequencing workflow:

   Prepare for your experiment

   You will need to:
   - Extract your DNA. Alternatively, start with whole cells
   - 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

   You will need to:
   - Amplify the genomic DNA from cells using the Qiagen REPLI-g UltraFast Kit
   - Tagment your DNA using the Fragmentation Mix in the kit
   - Attach sequencing adapters supplied in the kit to the DNA ends
   - Prime the flow cell, and load your DNA library into the flow cell

   

   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 a workflow for further analysis (this step is optional)

5. Important This protocol was developed using E. coli cells. For more information, please refer to the Qiagen REPLI-g UltraFast Kit protocol.
6. Important Compatibility of this protocol

   This protocol should only be used in combination with:

   • Rapid Sequencing Kit (SQK-RAD004)
   • R9.4.1 (FLO-MIN106) flow cells
     
   • Flow Cell Wash Kit (EXP-WSH004)

1. For this protocol, you will need >300 cells in 0.5 µl nuclease-free water, or 1-10 ng DNA in 1 µl nuclease-free water.
2. Input DNA

   How to QC your input DNA

   It is important that the input DNA meets the quantity and quality requirements. Using too little or too much DNA, or DNA of poor quality (e.g. highly fragmented or containing RNA or chemical contaminants) can affect your library preparation.

   For instructions on how to perform quality control of your DNA sample, please read the Input DNA/RNA QC protocol.

   Chemical contaminants

   Depending on how the DNA is extracted from the raw sample, certain chemical contaminants may remain in the purified DNA, which can affect library preparation efficiency and sequencing quality. Read more about contaminants on the Contaminants page of the Community.

3. Rapid Sequencing Kit contents

   

   Name	Acronym	Cap colour	No. of vials	Fill volume per vial (µl)
   Lambda DNA	LMD	Yellow	1	88
   Fragmentation Mix	FRA	Brown	1	30
   Rapid Adapter	RAP	Green	1	10
   Loading Beads	LB	Pink	1	360
   Sequencing Buffer	SQB	Red	1	300

4. Flow Cell Priming Kit contents (EXP-FLP002)

   

   Name	Acronym	Cap colour	No. of vials	Fill volume per vial (μl)
   Flush Buffer	FB	Blue	6	1,170
   Flush Tether	FLT	Purple	1	200

1. MinION Mk1B IT requirements

   Sequencing on a MinION Mk1B requires a high-spec computer or laptop to keep up with the rate of data acquisition. Read more in the MinION Mk1B IT Requirements document.

2. MinION Mk1C IT requirements

   The MinION Mk1C contains fully-integrated compute and screen, removing the need for any accessories to generate and analyse nanopore data. Read more in the MinION Mk1C IT requirements document.

3. 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.

4. Check your flow cell

   We highly recommend that you check the number of pores in your flow cell prior to starting a sequencing experiment. This should be done within 12 weeks of purchasing for MinION/GridION/PromethION or within four weeks of purchasing Flongle Flow Cells. Oxford Nanopore Technologies will replace any flow cell with fewer than the number of pores in the table below, when the result is reported within two days of performing the flow cell check, and when the storage recommendations have been followed. To do the flow cell check, please follow the instructions in the Flow Cell Check document.

   Flow cell	Minimum number of active pores covered by warranty
   Flongle Flow Cell	50
   MinION/GridION Flow Cell	800
   PromethION Flow Cell	5000

1. Input recommendation

   The REPLI-g UltraFast Mini Kit suggests an input of either 300 cells‡, or 1-10 ng‡‡ of gDNA. The method describing how to perform whole genome amplification differs, depending on sample type (cells or DNA), and details for each are provided. Please also refer to manufacturer's guides for further details.

   Notes:
   ‡ Although cells can be used in the REPLI-g kit, the chemical lysis step may not be sufficient to release the DNA from all cell types. Additional steps to ensure all cells are lysed may be required, depending on sample type.
   ‡‡ We have found that it is possible to reduce the input amount to, for example, 10 pg of gDNA. However, lowering the input may require the time of the amplification step to be increased. We recommend amplifying until the DNA concentration is >80 ng/μl (quantification performed using the Qubit dsDNA BR Assay Kit).

2. If starting with whole cells, follow the instructions below.

   Qiagen recommends that cells should be at a concentration of >600 cells/μl (please refer to manufacturer's guide for more information). If lower numbers of cells are used, please increase the amplification time until the concentration of amplified DNA is >80 ng/μl (quantification performed using the Qubit dsDNA BR Assay Kit).

3. In a 0.2 ml thin-walled PCR tube, mix together in the following order:

   Reagent	Volume
   Cell sample	0.5 µl
   Phosphate buffered saline (PBS)	1 µl
   Reagent D2 from the REPLI-g UltraFast Mini Kit	1.5 µl
   Total	3 µl

4. Incubate the reaction for 5 minutes at room temperature.
5. Add 1.5 µl Stop Reagent.
6. In a 0.2 ml thin-walled PCR tube, mix together in the following order:

   Reagent	Volume
   REPLI-g UltraFast Reaction Buffer	15 µl
   REPLI-g UltraFast Reaction Polymerase	1 µl
   Total	16 µl

7. Ensure the components are thoroughly mixed by pipetting, and spin down.
8. Add the lysed cells from the previous step to the tube.
9. Ensure the components are thoroughly mixed by pipetting, and spin down.
10. Incubate the reaction at 30° C until the DNA concentration is > 80 ng/µl. Quantification should be performed using the Qubit dsDNA BR Assay Kit.

    Typically this reaction would take ~2 hours, depending on the cell concentration and quality of the starting mateiral.

11. If starting with purified DNA, follow the instructions below.

    Qiagen recommends that 1-10 ng of gDNA, in a volume of 1 μl, should be put into the reaction (please refer to manufacturer's guide for more information). We have found that it is possible to reduce the input amount to 10 pg of gDNA. However, lowering the input may require a longer amplification time. We recommend amplifying until the DNA concentration is >80 ng/μl (quantification performed using the Qubit dsDNA BR Assay Kit).

12. Put the DNA in 1 μl into a clean tube.
13. Add 1 μl Buffer D1 to the tube. Mix by vortexing and spin down.
14. Incubate for 3 minutes at room temperature.
15. Add 2 μl Buffer N1 to the tube. Mix by vortexing and spin down.
16. In a 0.2 ml thin-walled PCR tube, mix together in the following order:

    Reagent	Volume
    REPLI-g UltraFast Reaction Buffer	15 µl
    REPLI-g UltraFast Reaction Polymerase	1 µl
    Total	16 µl

17. Ensure the components are thoroughly mixed by pipetting, and spin down.
18. Add the DNA from the previous step to the tube.
19. Ensure the components are thoroughly mixed by pipetting, and spin down.
20. Incubate the reaction at 30° C until the DNA concentration is > 80 ng/µl. Quantification should be performed using the Qubit dsDNA BR Assay Kit.

    Typically this reaction would take ~2 hours, depending on the cell concentration and quality of the starting mateiral.

21. Regardless of input material, continue with the protocol as follows:
22. In a 0.2 ml thin-walled PCR tube, mix the following:

    Reagent	Volume
    Amplified DNA	2.5 µl
    Nuclease-free water	5 µl
    Fragmentation Mix (FRA)	2.5 µl
    Total	10 µl

23. Ensure the components are thoroughly mixed by pipetting, and spin down.
24. Incubate the tube at 30°C for 1 minute and then at 80°C for 1 minute. Briefly put the tube on ice to cool it down.
25. Tip If heat blocks are used instead of a thermal cycler, incubation at both temperatures should be extended to 2 minutes.
26. Add 1 μl RAP to the 10 μl amplified DNA library.
27. Ensure the components are thoroughly mixed by pipetting, and spin down.
28. Incubate the reaction for 5 minutes at room temperature.
29. End of Step The prepared library is used for loading onto the flow cell. Store the library on ice until ready to load.

1. Thaw the Sequencing Buffer (SQB), Loading Beads (LB), Flush Tether (FLT) and one tube of Flush Buffer (FB) at room temperature before mixing the reagents by vortexing, and spin down at room temperature.
2. To prepare the flow cell priming mix, add 30 µl of thawed and mixed Flush Tether (FLT) directly to the tube of thawed and mixed Flush Buffer (FB), and mix by vortexing at room temperature.
3. 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

   

   

4. Optional Action

   Complete a flow cell check to assess the number of pores available before loading the library.

   This step can be omitted if the flow cell has been checked previously.

   See the flow cell check instructions in the MinKNOW protocol for more information.

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

   

6. Important Take care when drawing back buffer from the flow cell. Do not remove more than 20-30 µl, and make sure that the array of pores are covered by buffer at all times. Introducing air bubbles into the array can irreversibly damage pores.
7. After opening the priming port, check for a small air bubble under the cover. Draw back a small volume to remove any bubbles:
   1. Set a P1000 pipette to 200 µl
   2. Insert the tip into the priming port
   3. Turn the wheel until the dial shows 220-230 µl, to draw back 20-30 µl, or until you can see a small volume of buffer entering the pipette tip
      

   Note: Visually check that there is continuous buffer from the priming port across the sensor array.

   

8. Load 800 µl of the priming mix into the flow cell via the priming port, avoiding the introduction of air bubbles. Wait for five minutes. During this time, prepare the library for loading by following the steps below.

   

9. Thoroughly mix the contents of the Loading Beads (LB) tubes by vortexing.
10. Important The Loading Beads (LB) tube contains a suspension of beads. These beads settle very quickly. It is vital that they are mixed immediately before use.
11. In a new tube, prepare the library for loading as follows:

    Reagent	Volume per flow cell
    Sequencing Buffer (SQB)	34 µl
    Loading Beads (LB), mixed immediately before use	25.5 µl
    Nuclease-free water	4.5 µl
    DNA library	11 µl
    Total	75 µl

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

12. Complete the flow cell priming:
    1. Gently lift the SpotON sample port cover to make the SpotON sample port accessible.
    2. Load 200 µl of the priming mix into the flow cell priming port (not the SpotON sample port), avoiding the introduction of air bubbles.

    

    

13. Mix the prepared library gently by pipetting up and down just prior to loading.
14. Add 75 μl of the prepared library to the flow cell via the SpotON sample port in a dropwise fashion. Ensure each drop flows into the port before adding the next.

    

1. How to start sequencing

   Once you have loaded your flow cell, the sequencing run can be started on MinKNOW, our sequencing software that controls the device, data acquisition and real-time basecalling. For more detailed information on setting up and using MinKNOW, please see the MinKNOW protocol.

   MinKNOW can be used and set up to sequence in multiple ways:

   • On a computer either directly or remotely connected to a sequencing device.
   • Directly on a GridION, MinION Mk1C or PromethION 24/48 sequencing device.

   For more information on using MinKNOW on a sequencing device, please see the device user manuals:

   • MinION Mk1B user manual
   • MinION Mk1C user manual
   • GridION user manual

   ---

   To start a sequencing run on MinKNOW:

   1. Navigate to the start page and click Start sequencing.

   2. Fill in your experiment details, such as name and flow cell position and sample ID.

   3. Select the sequencing kit used in the library preparation on the Kit page.

   4. Configure the sequencing and output parameters for your sequencing run or keep to the default settings on the Run configuration tab.

   Note: If basecalling was turned off when a sequencing run was set up, basecalling can be performed post-run on MinKNOW. For more information, please see the MinKNOW protocol.

   5. Click Start to initiate the sequencing run.

2. Data analysis after sequencing

   After sequencing has completed on MinKNOW, the flow cell can be reused or returned, as outlined in the Flow cell reuse and returns section.

   After sequencing and basecalling, the data can be analysed. For further information about options for basecalling and post-basecalling analysis, please refer to the Data Analysis document.

   In the Downstream analysis section, we outline further options for analysing your data.

1. After your sequencing experiment is complete, if you would like to reuse the flow cell, please follow the Flow Cell Wash Kit protocol and store the washed flow cell at 2-8°C.

   The Flow Cell Wash Kit protocol is available on the Nanopore Community.

2. Tip We recommend you to wash the flow cell as soon as possible after you stop the run. However, if this is not possible, leave the flow cell on the device and wash it the next day.
3. Alternatively, follow the returns procedure to flush out the flow cell ready to send back to Oxford Nanopore.

   Instructions for returning flow cells can be found here.

   Note: All flow cells must be flushed with deionised water before returning the product.

4. Important If you encounter issues or have questions about your sequencing experiment, please refer to the Troubleshooting Guide that can be found in the online version of this protocol.

1. Post-basecalling analysis

   There are several options for further analysing your basecalled data:

   1. EPI2ME workflows

   For in-depth data analysis, Oxford Nanopore Technologies offers a range of bioinformatics tutorials and workflows available in EPI2ME. The platform provides a vehicle where workflows deposited in GitHub by our Research and Applications teams can be showcased with descriptive texts, functional bioinformatics code and example data.

   2. 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.

   3. 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 resource centre and search for bioinformatics tools for your application. 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.

1. Below is a list of the most commonly encountered issues, with some suggested causes and solutions.

   We also have an FAQ section available on the Nanopore Community Support section.

   If you have tried our suggested solutions and the issue still persists, please contact Technical Support via email (support@nanoporetech.com) or via LiveChat in the Nanopore Community.

2. Low sample quality

   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 document. Please try an alternative extraction method that does not result in contaminant carryover. Consider performing an additional SPRI clean-up step.
   Low RNA integrity (RNA integrity number <9.5 RIN, or the rRNA band is shown as a smear on the gel)	The RNA degraded during extraction	Try a different RNA extraction method. For more info on RIN, please see the RNA Integrity Number document. Further information can be found in the DNA/RNA Handling page.
   RNA has a shorter than expected fragment length	The RNA degraded during extraction	Try a different RNA extraction method. For more info on RIN, please see the RNA Integrity Number document. Further information can be found in the DNA/RNA Handling page. We recommend working in an RNase-free environment, and to keep your lab equipment RNase-free when working with RNA.

3. 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.
   Low recovery after end-prep	The wash step used ethanol <70%	DNA will be eluted from the beads when using ethanol <70%. Make sure to use the correct percentage.

1. Below is a list of the most commonly encountered issues, with some suggested causes and solutions.

   We also have an FAQ section available on the Nanopore Community Support section.

   If you have tried our suggested solutions and the issue still persists, please contact Technical Support via email (support@nanoporetech.com) or via LiveChat in the Nanopore Community.

2. Fewer pores at the start of sequencing than after Flow Cell Check

   Observation	Possible cause	Comments and actions
   MinKNOW reported a lower number of pores at the start of sequencing than the number reported by the Flow Cell Check	An air bubble was introduced into the nanopore array	After the Flow Cell Check it is essential to remove any air bubbles near the priming port before priming the flow cell. If not removed, the air bubble can travel to the nanopore array and irreversibly damage the nanopores that have been exposed to air. The best practice to prevent this from happening is demonstrated in this video.
   MinKNOW reported a lower number of pores at the start of sequencing than the number reported by the Flow Cell Check	The flow cell is not correctly inserted into the device	Stop the sequencing run, remove the flow cell from the sequencing device and insert it again, checking that the flow cell is firmly seated in the device and that it has reached the target temperature. If applicable, try a different position on the device (GridION/PromethION).
   MinKNOW reported a lower number of pores at the start of sequencing than the number reported by the Flow Cell Check	Contaminations in the library damaged or blocked the pores	The pore count during the Flow Cell Check is performed using the QC DNA molecules present in the flow cell storage buffer. At the start of sequencing, the library itself is used to estimate the number of active pores. Because of this, variability of about 10% in the number of pores is expected. A significantly lower pore count reported at the start of sequencing can be due to contaminants in the library that have damaged the membranes or blocked the pores. Alternative DNA/RNA extraction or purification methods may be needed to improve the purity of the input material. 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.

3. MinKNOW script failed

   Observation	Possible cause	Comments and actions
   MinKNOW shows "Script failed"		Restart the computer and then restart MinKNOW. If the issue persists, please collect the MinKNOW log files and contact Technical Support. If you do not have another sequencing device available, we recommend storing the flow cell and the loaded library at 4°C and contact Technical Support for further storage guidance.

4. 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.

5. Shorter than expected read length

   Observation	Possible cause	Comments and actions
   Shorter than expected read length	Unwanted fragmentation of DNA sample	Read length reflects input DNA fragment length. Input DNA can be fragmented during extraction and library prep. 1. Please review the Extraction Methods in the Nanopore Community for best practice for extraction. 2. Visualise the input DNA fragment length distribution on an agarose gel before proceeding to the library prep. In the image above, Sample 1 is of high molecular weight, whereas Sample 2 has been fragmented. 3. During library prep, avoid pipetting and vortexing when mixing reagents. Flicking or inverting the tube is sufficient.

6. Large proportion of unavailable pores

   Observation	Possible cause	Comments and actions
   Large proportion of unavailable pores (shown as blue in the channels panel and pore activity plot) The pore activity plot above shows an increasing proportion of "unavailable" pores over time.	Contaminants are present in the sample	Some contaminants can be cleared from the pores by the unblocking function built into MinKNOW. If this is successful, the pore status will change to "sequencing pore". If the portion of unavailable pores stays large or increases: 1. A nuclease flush using the Flow Cell Wash Kit (EXP-WSH004) can be performed, or 2. Run several cycles of PCR to try and dilute any contaminants that may be causing problems.

7. Large proportion of inactive pores

   Observation	Possible cause	Comments and actions
   Large proportion of inactive/unavailable pores (shown as light blue in the channels panel and pore activity plot. Pores or membranes are irreversibly damaged)	Air bubbles have been introduced into the flow cell	Air bubbles introduced through flow cell priming and library loading can irreversibly damage the pores. Watch the Priming and loading your flow cell video for best practice
   Large proportion of inactive/unavailable pores	Certain compounds co-purified with DNA	Known compounds, include polysaccharides, typically associate with plant genomic DNA. 1. Please refer to the Plant leaf DNA extraction method. 2. Clean-up using the QIAGEN PowerClean Pro kit. 3. Perform a whole genome amplification with the original gDNA sample using the QIAGEN REPLI-g kit.
   Large proportion of inactive/unavailable pores	Contaminants are present in the sample	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.

8. Reduction in sequencing speed and q-score later into the run

   Observation	Possible cause	Comments and actions
   Reduction in sequencing speed and q-score later into the run	For Kit 9 chemistry (e.g. SQK-LSK109), fast fuel consumption is typically seen when the flow cell is overloaded with library (please see the appropriate protocol for your DNA library to see the recommendation).	Add more fuel to the flow cell by following the instructions in the MinKNOW protocol. In future experiments, load lower amounts of library to the flow cell.

9. Temperature fluctuation

   Observation	Possible cause	Comments and actions
   Temperature fluctuation	The flow cell has lost contact with the device	Check that there is a heat pad covering the metal plate on the back of the flow cell. Re-insert the flow cell and press it down to make sure the connector pins are firmly in contact with the device. If the problem persists, please contact Technical Services.

10. Failed to reach target temperature

    Observation	Possible cause	Comments and actions
    MinKNOW shows "Failed to reach target temperature"	The instrument was placed in a location that is colder than normal room temperature, or a location with poor ventilation (which leads to the flow cells overheating)	MinKNOW has a default timeframe for the flow cell to reach the target temperature. Once the timeframe is exceeded, an error message will appear and the sequencing experiment will continue. However, sequencing at an incorrect temperature may lead to a decrease in throughput and lower q-scores. Please adjust the location of the sequencing device to ensure that it is placed at room temperature with good ventilation, then re-start the process in MinKNOW. Please refer to this FAQ for more information on MinION temperature control.

11. 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

12. 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.

13. 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.