Quick Guide to mNGS Analysis

Overview

Here we describe general steps to perform metagenomic (mNGS) analysis within CZ ID. See Guide to mNGS Data Analysis for a detailed description of how to approach mNGS analyses. 

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mNGS Analysis Steps

To perform mNGS analysis:

1) Log in to your CZ ID account.

2) Go to the Upload page from the Discovery page by selecting the "Upload" link by your username. Follow prompts to upload sequencing FASTQ files, add metadata, and review. For detailed step-by-step guides see:

• Uploading mNGS Illumina Data
• Uploading mNGS Nanopore Data

1. Upload Page: Click link to navigate to the Upload page.
2. Upload Steps: The upload process is divided into three general sections (Samples, Metadata, and Review). The current section will be highlighted in blue.

3) Go to the Project page after uploading samples to view sample run status. The sample run should be listed under the Metagenomis tab if you uploaded short-read data (Illumina) or the Metagenomics - Nanopore tab if you are working with long reads.

1. Project Name: Indicates you are viewing the project's page.
2. Metagenomics Tabs: Select the appropriate tab for short-read (Metagenomics) and long-read (Metagenomics - Nanopore) samples.
3. Run Status: Indicates if the run has been completed, completed with issue, or failed.

4) Once the sample run is completed, click on a sample row to view its Sample Report. You can sort and filter the report table to focus on relevant identified taxa. See the following links for detailed information about sample reports and filtering:

• Short-read data: Illumina Sample Report and Filter Feature
• Long-read data: Nanopore Sample Report and Filter Feature

1. Filter Options: Filter the report table based on categories (e.g., bacteria, viruses) or metric thresholds (e.g., rPM 100) to focus on relevant results.
2. Metrics per Taxa: Reported metrics for matches found in the NT and NR databases including: Score, Z-score, reads per million (rPM) or bases per million (bPM), # of reads (r) or bases (b), # of contigs, # of reads or bases assembled into contigs (contig r or contig b), average % identity of alignments (% id), average length of alignments (L), and E-value. Sample reports for long-read data include metrics at the base level and do not include Score and Z-score values.
3. Annotation Tag: Use tags to note if identified taxa are a "hit", "no hit", or "inconclusive".
4. Details at the Species Level: By default, results are shown at the genus level. However, results at the species level can be viewed by clicking on the downward arrow located by genus names.
5. Analysis Icons: Icons become visible when hovering over a taxon name. Use analysis icons to view coverage, run BLAST, build trees, generate viral consensus genomes, or download sequences associated with the taxon. Note that BLAST, phylogenetic tree, and viral consensus genome icons are only available for short-read data.
6. Known Pathogen Flag: Flags highlight pathogenic taxa.

5) Confirm identified taxa by evaluating genome coverage and using BLAST.

Short-read sequencing data (Illumina):

• • View coverage for identified taxa
  • Use BLAST

Long-read sequencing data (Nanopore):

• • View coverage for identified taxa
  • Use BLAST

6) If you are working with short-reads, you can perform various analysis from the Sample Report page, including:

• Apply background models to account for contamination
• Assemble consensus genomes
• Create heatmaps
• Build phylogenetic trees

7) Download reports and data of interest, including intermediate files generated throughout the pipeline.

• Download data from mNGS Illumina Project and Sample Report pages
• Download Nanopore data