Metagenomic Analysis in Geneious Prime

This metagenomic analysis series shows how to process NGS amplicon data in Geneious Prime, from preprocessing with BBDuk trimming and read merging, through clustering reads into OTUs and BLASTing them against the NCBI 16S microbial database, to building a custom taxonomy database and classifying sequences with the Sequence Classifier. The series spans four videos, 4 to 5 minutes each.

2. Cluster and Batch Blast Amplicon Data
4. Classifying Amplicon Data

1. Preprocessing of NGS Amplicon Data

Trim paired Illumina amplicon reads with BBDuk, removing adapters and low-quality bases, then merge overlapping pairs into consensus sequences. Use the Length Graph to extract reads within the expected size range and exclude contaminated sequences.

LENGTH 5 minutes

2. Cluster and Batch Blast Amplicon Data

Cluster amplicon reads into OTUs using de novo assembly with high-stringency settings, since BLASTing millions of individual reads isn't practical. BLAST one representative sequence per OTU against a local NCBI 16S database to build a reference.

LENGTH 5 minutes

3. Creating a Taxonomy Database

Format BLAST hits into a taxonomy database for the Sequence Classifier: remove duplicates, download full sequences, extract only the BLAST hit regions, then batch rename each sequence to its source organism using the per-sequence metadata field.

LENGTH 4 minutes

4. Classifying Amplicon Data

Classify preprocessed, clustered amplicon reads against your taxonomy database using the Sequence Classifier, setting overlap identity cutoffs per taxonomic level, such as 95% for genus. Review results in the Summary, Classifications, and Results tables.

LENGTH 4 minutes

Recommended Resources

Plugin - BBDuk Trimmer

Download the plugin for quality trimming and filtering your sequences.

How to preprocess NGS reads?

The best practice for preprocessing NGS reads in Geneious Prime.

Manual for Sequence Classifier

A guide to using the Sequence Classifier plugin in Geneious Prime.

De novo assembly advisor decision tree & validation table

Identify the most appropriate assembly algorithm for your data.

More Geneious Academy

TUTORIALHow to Analyze a Metagenomic Dataset in Geneious Prime

Assemble, filter and classify a 16S rRNA V4 amplicon dataset from paired Illumina MiSeq reads.

Read Tutorial

VIDEO SERIESDe Novo Assembly in Geneious Prime

Learn to assemble NGS reads without a reference genome, covering sensitivity settings, read normalization, and circular genome assembly in Geneious Prime.

Watch Series