Upload Nanopore Data

Overview

Here we list steps for uploading data to the mNGS Nanopore pipeline. After uploading data and the pipeline run is completed you can analyze data to identify microbes of interest and download results. Click here to learn about the mNGS Nanopore pipeline. 

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Upload Data

CZ ID only accommodates one sequence file per sample when uploading Nanopore sequencing data to the pipeline. Multiple FASTQ files for a given sample will be automatically concatenated if filenames follow a certain format (see Automatic Concatenation of Nanopore Files for details). If there are multiple FASTQ files per sample and filenames do not follow the format recognized by the platform, you need to concatenate (or combine) them into a single file before upload.

To upload Nanopore sequencing data to your CZ ID account:

1) Log in to your CZ ID account.

2) Navigate to the Upload Samples page.

Page for uploading samples. Note that the upload process is divided into three general sections (Samples, Metadata, and Review).

3) Select or create a project within the Upload Samples page. When creating a new project, you will need to add a project name, select if the project will be public within CZ ID or private, and provide a project description.

If you are creating a new project, fill in the information requested within the New Project dialog box. After adding your project information, the “Create Project” button will be enabled. Click the button to save the new project.

4) The next step within the Upload Samples page is to select the analysis type. Under “Analysis Type”, select “Metagenomics” and check the Nanopore box as the sequencing platform. You will be prompted to specify the Guppy basecaller model used to generate the FASTQ files. 

 

Guppy basecaller

The Guppy basecaller model will determine steps used for assembly based on the expected sequence error rate. The Guppy basecaller options include: fast, high accuracy (hac), and super accuracy (super) models. We highly recommend using the “super” model during basecalling whenever possible. The “super” model has been shown to reduce sequence error rates compared to the other two models. If you don’t have access to the “super” model, the “hac” model can reduce error rates by ~ 2% relative to the fast model (see research article). The “fast” model should be avoided if possible.

5) The final step within the Upload Samples page is to upload sequence files. Upload FASTQ (“.fastq” or “.fq”) or compressed FASTQ (“.fastq.gz” or “.fq.gz”) files directly from your computer by dragging and dropping files into the provided box or using your file browser. Click here if you need to upload FASTA files.

Note: Make sure to concatenate sequencing files into a single file prior to uploading sequences if there are multiple FASTQ files per sample that do not follow the format recognized by the platform (see Automatic Concatenation of Nanopore Files for details).

Click “Continue” after providing project information, specifying analysis type, and uploading sequence files.

Filenames will be used to name samples automatically. Note the following when naming your sequence files:

• • Filenames must be no longer than 120 characters
  • Filenames can only contain letters from the English alphabet (A-Z, upper and lower case), numbers (0-9), periods (.), hyphens (-) and underscores (_)
  • Spaces are not allowed

6) Continue to the next section, Upload Metadata.

7) Fill out the appropriate metadata. Note that there are six required metadata fields, including: Host Organism, Sample Type, Water Control, Nucleotide Type, Collection Date, and Collection Location. See Adding Metadata for details. You can enter metadata manually or upload a metadata file in comma-delimited format (".csv” file extension).

For manual entry, stay on the “Manual Input” tab (default). Fill in metadata information using the provided fields directly through the web interface. After entering information for all the required fields, continue to the next section (Review).

Prepare a metadata file locally on your computer by downloading a metadata template or by copying and pasting required metadata fields into a file. Save your metadata as a “comma-delimited” file and upload the file under the “CSV Upload” tab. If there are no errors, continue to the next section (Review).

8) Continue to the next section, Review.

9) Review the project, sample, and analysis information. Accept the CZ ID Privacy Policy and Terms of Service and click “Start Upload”.

Start upload after reviewing submission information and accepting CZ ID Terms of Service.

You will see a popup indicating the status of your sample upload. Once upload is completed, click “Go to Project” to view the status of your samples.

10) Go to the Project page to view sample status and analyze results.