Upload or Select Data for AMR Analysis

Jump to Section:

• Upload Data
• Select mNGS Samples

Overview

Here we describe how to kick off your antimicrobial resistance (AMR) analysis within CZ ID. There are two options for submitting data for AMR analysis. You can upload a new dataset to your CZ ID account or you can select metagenomic (mNGS) samples already in your account.

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Upload Data for AMR Analysis

Follow the steps listed below to upload data for AMR analysis.

• Step 1: Go to Upload Page
• Step 2: Specify Project
• Step 3: Select Analysis Type
• Step 4: Select Sequencing Files
• Step 5: Add Metadata
• Step 6: Review

Step 1: Go to Upload Page

Log in to CZ ID using your email and password. Once logged in, you will see your name in the upper right-hand corner of the application. You will see a link to the Upload page next to your username.

1. Upload Page Link: Click this link to open the Upload page.
2. Upload Steps: Upload is divided into three general stages to upload samples ("Samples"), add metadata ("Metadata"), and review the information ("Review"). The current stage will be highlighted in blue. 

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Step 2: Specify Project

Select or create a project within the Select Samples page. The project selection will affect the pipeline and database version used to run the samples given that the pipeline version for all analysis types and NCBI Index Date are assigned upon project creation (see Pipeline Version and NCBI Index Date for details). Therefore, all samples within a project will run on the same major pipeline version and use the same NCBI Index Date. 

1. Create New Project: Use this link to create a new project. A dialog box will appear to enter the new project information.
2. Project Dropdown Menu: Use the dropdown menu to upload samples to an existing project.

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. Click the Create Project button to finish creating the new project.

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Step 3: Select Analysis Type

Under “Analysis Type”, select Antimicrobial Resistance. The AMR pipeline version that will be used to run the samples will be specified here.

You can choose to run metagenomic analysis ("Metagenomics") in parallel to the AMR run. To do this, select Metagenomics and Antimicrobial Resistance under the Analysis Type section. Note that the AMR pipeline is only available for short-read data (Illumina). If you select the Metagenomics option, you will see the pipeline version and NCBI Index Date that will be used to run the metagenomic analysis.

1. Sequencing Platform Options: When running mNGS analysis in addition to AMR, you can only select the short-read (Illumina) platform. 
2. Pipeline Version: Specifies the mNGS Illumina pipeline version that will be used to run samples.
3. NCBI Index Date: Specifies the date nucleotide (NT) and protein (NR) databases used to analyze samples were downloaded from NCBI.

Pipeline Version

The pipeline version that will be used to run uploaded samples can be seen once you select a project and analysis type (i.e. Antimicrobial Resistance). CZ ID uses a three-level pipeline versioning system where the first number indicates the major pipeline version followed by numbers that specify minor version and patch updates. For example, pipeline v1.2.15 refers to major pipeline version 1, minor pipeline version 2, and patch version 15.  

The project’s pipeline version will be automatically assigned upon project creation based on the latest version available for each analysis type. This pipeline version pinning by project helps to ensure that all sample runs within a project are comparable. For example, if your project is pinned to AMR pipeline v1.2.15, all new samples uploaded to that project will run on major AMR pipeline v1. This system enables minor pipeline updates to be associated with the same major version while still allowing your results to be comparable. 

You will see a Warning Icon ( ) if there is a new major pipeline version available. To use this new pipeline, you must create a new project.

Notes regarding pipeline versioning:

• Projects created before February 08, 2024  may include multiple major pipeline versions.
• The AMR pipeline version will dictate which version of the Comprehensive Antibiotic Resistance Database (CARD) will be used to detect AMR genes. Samples uploaded to AMR pipeline v1.2.4 and later will be compared against the latest CARD version available in CZ ID, whereas samples uploaded to prior versions will use CARD v3.2.3. Click here for details regarding CARD version updates.
• The AMR pipeline uses the same QC and host filtering as the mNGS pipeline and, thus, changes to the mNGS pipeline also affect AMR analysis. Click here to learn about QC and host filtering step differences between major pipeline versions.
• Metagenomic samples selected for AMR analysis (see section below):
  • Samples run on mNGS pipeline v8.0 (and later) that are selected for AMR analysis will be compared against the latest CARD.
  • Samples run on pipeline versions prior to mNGS pipeline v8.0 that are selected for AMR analysis will be compared against CARD v3.2.3.

NCBI Index Date

The NCBI Index that will be used to process uploaded samples can be seen if you select Metagenomics - Illumina, in addition to AMR, for the analysis type. The NCBI Index Date indicates the date the NCBI NT and NR databases were downloaded for use by CZ ID. This index date can be used to find associated GenBank release numbers (see GenBank release notes). The downloaded databases are then compressed and indexed by CZ ID. Newer versions of the index will have the most up to date taxon information from NCBI.

The project’s NCBI Index Date will be automatically assigned upon project creation based on the latest version available. Each project is pinned to one NCBI Index Date to ensure that all sample runs within a project are comparable*. For example, if your project is pinned to NCBI Index Date 2021-01-22, all new samples uploaded to that project will run on Index 2021-01-22. 

You will see a Warning Icon ( ) if there is a new NCBI Index available. To use this new Index, you must create a new project.

*Note: Projects created before February 08, 2024 may include multiple NCBI Index Dates.

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Step 4: Select Sequencing Files

After specifying the analysis type, scroll down to "Select Files" to upload FASTQ (“.fastq” or “.fq”) or compressed FASTQ (“.fastq.gz” or “.fq.gz”) files directly from your computer or BaseSpace account. Click here if you have FASTA files.

Upload Files from Your Computer

Select Your Computer tab to upload files directly from your computer.

1. Your Computer Tab: Use this tab (default) to select sequencing files found in your computer. 
2. Upload Box: Drag and drop files into the provided box or click the link to use your file browser.
3. Sample List: Sequencing files ready for upload will be listed here. Sample names will be based on the sequence filenames (see file requirements).
4. Continue Button: After selecting files, use this button to continue to the Upload Metadata section.

If you have sequencing files split over multiple lanes per sample, CZ ID will automatically detect files representing the same sample based on Illumina's naming convention and concatenate these files for you. For example, if you were to upload one paired-end sequence sample split over three lanes, such sample would have six files. In the screenshot below you can see that CZ ID automatically detects that each file is part of the same sample. 

Upload Files from BaseSpace

If your files are hosted on BaseSpace, you can pull them directly into CZ ID. Select the BaseSpace tab under the Select Files section to access your files. Click the Connect to BaseSpace button to launch the BaseSpace login page. 

Use your credentials to log in to BaseSpace and select files for upload. 

Once you have selected and reviewed the files you want to process, click the Continue button at the bottom of the screen to continue to the next step (Upload Metadata). 

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Step 5: Add Metadata

Add the appropriate sample metadata through the Upload Metadata page. 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).

Manual Metadata Entry

Use 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, click the Continue button to go to the Review section.

1. Manual Input Tab: Use this tab (default) when entering metadata directly throught the web interface.
2. Metadata Table: Enter information for each column or field. By default, the required fields will be listed on the table. You can add additional columns through the Metadata Dropdown Menu. 
3. Metadata Dropdown Menu: Click the plus sign to view and add optional metadata fields to the Metadata Table.

Upload Metadata File 

Use the "CSV Upload" tab to upload a comma-separated value (CSV) file with metadata. If there are no errors, click the Continue button to go to the Review section.

1. CSV Upload Tab: Select this tab to upload a metadata file.
2. Metadata Template: Click template link to download a CSV file that will be already populated with sample names and metadata fields. Edit the file to include the appropriate metadata and save it. You are not required to use the provided template.
3. Metadata File Upload Box: Use this box to upload the metadata file.

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Step 6: Review

Use the Review page to review the project, sample, and analysis information. The "Edit" links by each section can be used to edit project and sample information if you need to correct anything before upload. After reviewing sample and metadata information, please accept CZ ID's Terms of Service and Data Privacy Policy. Press Start Upload to begin the upload process to our server and kick off the analysis pipeline. 

Note regarding host filtering:

"Host Subtraction" information is located below the table listing samples to be uploaded. This information tells you how your selection of host organism will affect the pipeline, specifically the host subtraction step. If CZ ID has the genome of the host organism, the pipeline will subtract out reads aligning to that genome. Regardless of your choice of host, the pipeline will always remove ERCCs and reads aligning to the Human genome (reference: HG38 and T2T-CHM13 assemblies). If CZ ID does not have the genome that matches to your host organism, you can request it by following the instructions in our FAQs.

After pressing Start Upload, you will see a modal showing the upload progress. DO NOT close the web page while the upload is in progress. Otherwise, the upload will be canceled and you will have to start your upload over. 

Wait until you will see an "Uploads completed!" confirmation message confirming that your samples have been uploaded successfully. Once you see the confirmation message you can close your window or press "Go to Project" to navigate to the Project page where you can view sample status and analyze results.

You can see the status of your run by going to the Project Page. The image below highlights features of a Project page listing the status of AMR samples. Once the AMR pipeline run is completed, you can view the AMR Sample Report and download results.

1. Project Name
2. Antimicrobial Resistance Tab
3. Sample Status: Specifies sample progress. When the run is successfully completed, you will see a "Complete" status highlighted in green.

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Select mNGS Samples for AMR Analysis

You can perform AMR analyses on samples that have already run through the short read (Illumina) mNGS pipeline. This feature is only available for mNGS samples run on Illumina mNGS pipeline v5.0 and later.

Steps to Run AMR Analysis on mNGS Samples

1. Go to the Metagenomics tab found within the Project page of interest.
2. Select mNGS sample(s) for AMR analysis.
3. Click on the More Actions icon.
4. Select "Run Antimicrobial Resistance Pipeline" from the dropdown menu to start AMR analysis on selected samples.

   You will see a confirmation or warning message on the top right-hand corner of the page. If everything is working well, you will see a popup message highlighted in blue indicating that samples are running on the Antimicrobial Resistance pipeline. You will see a popup message highlighted in yellow if the AMR analysis failed to launch. Make sure you are not trying to run samples that have been already submitted for AMR analysis.

5. Go to the Antimicrobial Resistance tab
6. Check the status of the AMR run.
7. Once the AMR pipeline run is completed, click on the sample to view the AMR Sample Report and/or download results.