Starting Metagenomics Experiments

The NHS RMg platform runs separately to the ONT sequencing software. Users should perform steps in the following order:

1. Run a flow cell check.
2. Start the sequencing experiment using the relevant ONT software.
3. Launch the NHS RMg platform analysis.
4. Check the NHS_RMg_platform/results directory for reports.

Running a flow cell check MinKNOW

A flow cell check should always be performed before starting a run. Starting a run with sub-optimal pore availability can produce invalid results. Oxford Nanopore Technologies will replace any flow cell that falls below the warranty number of active pores within three months of purchase, provided that you report the results within two days of performing the flow cell check and you have followed the storage recommendations. A MinION flow cell (used also in the GridION) should have > 800 active pores.

1. Select 'Flow Cell Check' from the MinKNOW 'Start' screen.

2. Indicate the corresponding sequencing positions/flow cells you'd like to check by selecting the square icons below the 'Flow Cell Check' title.

3. Click on the green Start button and wait for the flow cell check to complete.

Starting a sequencing experiment using MinKNOW

1. From the Start screen in MinKNOW select 'Start Sequencing'.

2. Select the position occupied by the flow cell loaded for the sequencing experiment, enter the Experiment and Sample IDs.

3. Select 'Continue to kit selection' at the bottom of the screen.

4. Select the SQK-RPB004 kit from the Kit selection screen. Click 'Continue to run options' at the bottom of the window.

5. In the run options screen, set the sequencing experiment to last for 24 hours. Leave the read length at 200 bp and select 'Continue to analysis' at the bottom of the window.

6. On the Analysis window, under barcoding, select 'Edit options'. In the popup window, select ‘Trim Barcodes’ and 'Barcode at both ends'. Select the 'Continue to output' button at the bottom of the window.

7. On the Output window, deselect the FAST5 option. Where the FASTQ checkbox is selected, click the gear icon and set 'Reads per file' to 100.

8. Continue to the 'final review' section and confirm the parameters match the table below:

Parameter	Value
Selected Kit	SQK-RPB004
Run length	24 hours
Minimum read length	200 bp
Adaptive sampling	Off
Basecalling	On (High accuracy basecalling)
Barcoding	On
Require both ends	On
Detect mid-read barcodes	On
Alignment	Off
Location	/data
FAST5	Off
FASTQ	On (Gzip, 100 reads per file)
Read filtering	Qscore:10 Readlength: unfiltered, Read splitting: Disabled

9. Start the sequencing experiment. After the flow cell reaches temperature, navigate to the barcodes screen to verify data output.

\pagebreak

Running the Metagenomics Workflow MinKNOW

The Metagenomics Launcher is a tool designed to acquire sample metadata and initiate the analysis of sequencing data to populate PDF/HTML reports. Reports are generated every 0.5, 1, 2, 16 and 24 hours, stored in the ./NHS_RMg_platform/reports/{Lab/SampleID} directory.

1. Double click the Metagenomics Launcher icon on the GridION desktop, the CIDR Metagenomics Launcher should appear alongside a terminal window.

2. Select the number of samples to be analysed from the ‘Select number of rows’ dropdown (2).

3. Fill out the ‘Experiment ID’ field (3) with an appropriate identifier for the batch of samples. This field will be used to ease the identification runs in downstream analyses.

4. Populate the launcher fields by either loading an existing sample sheet (see Additional Information for guide), or manually populate fields 4 to 14.

5. Where appropriate, run the Anonymisation Functions (15). See the 'Anonymisation' section for more details on set up and usage.

6. Click on Launch pipeline and click ‘OK to start the analysis’. The window will then freeze until the workflow process has completed. After a minute, the terminal window accompanying the workflow launcher should start displaying log outputs from the workflow.

7. Approximately 10-15 minutes after the sequencing timepoint, reports will be published to \ ./NHS_RMg_platform/reports/{Lab/SampleID}/

8. For any errors, missing reports or queries, please go to the Troubleshooting section.

Running a flow cell check Gourami

A flow cell check should always be performed before starting a run. Starting a run with sub-optimal pore availability can produce invalid results. Oxford Nanopore Technologies will replace any flow cell that falls below the warranty number of active pores within three months of purchase, provided that you report the results within two days of performing the flow cell check and you have followed the storage recommendations. A MinION flow cell (used also in the GridION) should have > 800 active pores.

1. From the Gourami ‘Overview’ page, select ‘View Flow Cell’ at the appropriate flow cell position.

2. From the flow cell information screen, select ‘Run flow cell check’ on the right-hand side (see Figure 3).

3. If the pore count is < 800 and the flow cell is still in warranty, contact ONT for a replacement within two days of completing the check.

\pagebreak

Starting a sequencing experiment and initiating metagenomics analysis Gourami

1. A video guide is available on the Network Hub website for this procedure.

2. Visit the 'Limitations' section below and FAQs on the Network Hub for an up-to-date description of issues and solutions affecting the workflow results.

3. Functions referred to in this section will have numeric tag in brackets matching its location on the diagram below. A diagram mapping features is available in the Technical Information section.

4. Open Gourami by clicking on the ONT icon in the taskbar to the left of the screen or on the desktop.

5. In the ‘Run Setup’ section, select “RPB004 prerelease” and press continue.

3. Select the position of the flow cell intended for the sequencing experiment. A flow cell check will be required if not completed already.

The 'RPB004-prerelease' option may appear differently on your device. Consult with an ONT rep if this option is missing from your device.

4. Switching applications, open the Metagenomics Launcher from the desktop icon. Fill out the the fields on the launcher in the order listed below:

• Number of rows (2)
• Experiment ID (3)
• ONT Barcode (6)
• Lab/Sample ID (7)

5. Click on the 'Generate Gourami sample sheet' button (17).

6. Switching back to Gourami, import the generated sample sheet. This can be found within the NHS RMg Metagenomics Platform environment at the following path: NHS_RMg_platform/sample_sheets/gourami/{yyyy_mm_dd@hh_mm_ss_{Experiment ID}. Validate the entries and click 'Continue'.

7. Prime and load the flow cell as per the relevant laboratory SOP and commence the sequencing experiment.

8. Wait until the the sequencing experiment has started and at least one barcode/sample status has switched to the green 'Found' status.

\pagebreak

9. Switch back to the Metagenomics Launcher, click on the 'Refresh Directories' button (21) and populate the remaining fields 8-14.

Clicking the black triangle to the right of selected column headers will clone the value in row 1 to all below.

10. Where appropriate, run the Anonymisation Functions (15). See the Anonymisation section for more details on set up and usage.

11. Click on Launch pipeline and click ‘OK to start the analysis’. The window will then freeze until the workflow process has completed. After a minute, the terminal window accompanying the workflow launcher should start displaying log outputs from the workflow.

12. Approximately 10-15 minutes after the sequencing timepoint, reports will be published to \ ./NHS_RMg_platform/reports/{Lab/SampleID}/

• Reports usually take between 10 - 20 minutes to publish after data acquisition timepoints elapse.
• The workflow generates reports at five timepoints: 0.5, 1, 2, 16, and 24 hours.
• For real-time sequencing runs, the workflow waits for each timepoint to elapse before publishing the corresponding report, spanning a 24-hour period.
• For retrospective analysis of existing datasets, reports are generated immediately for the same timepoints based on when the data was originally acquired, without waiting between timepoints.

For aborted runs, crashes, mislabelling, use the 'Force overwrite' (18) function on the Launcher to delete and replace analysis outputs (results and reports) with a new run. Use this also if a "directory locked" error is observed.

Any data deleted by this function will be stored in the ./NHS_RMg_platform/recycle_bin directory.

Do not close the terminal or the Launcher window until all reports have been published or the pipeline has stopped operation.

For any errors, missing reports or queries, please go to the Troubleshooting section.