Nephele User Guide

Nephele Pipelines


Nephele provides QIIME1, mothur and DADA2 pipelines for amplicon data and the bioBakery pipeline for metagenome shotgun data. In addition, we have added a sequence data quality check pipeline so that you can inspect and control for your data quality before analysis.

We also like learning about new and different pipelines that could better serve your research and educational needs. If you have a suggestion of a tool or analysis for Nephele, please fill out this form.
*Note: your request will be considered in planning new features, but it doesn’t guarantee implementation.

Data Types

Nephele v2 supports demultiplexed paired-end and single-end FASTQ files. Please see the supported data type per pipeline below.

If you need help figuring out what type of data you have, please see the FAQ.

Pre-processing Quality Check (QC) Pipeline

Nephele provides a pre-processing quality check pipeline for demultiplexed paired-end and single-end FASTQ files. Please see this FAQ on why you may want to run QC pipeline before you run a microbiome analysis. The Nephele QC pipeline can run a quality control check (FastQC), Trim primers and/or adapters, Trim and/or Filter reads based on quality scores, Merge read pairs, and provides summary graphs of the QC steps.

The features list below is a summary of the Nephele QC pipeline workflow.

Pipeline Features Pre-processing Quality Check
FastQC sequence quality check
Always run
Trim primers and/or adapters
Run if selected
Trim reads based on quality scores
Run if selected
Filter reads based on quality scores
Run if selected
Merge read pairs
Run if selected
Summary graphs of QC steps
Always run

* Please note that Trim primers and/or adapters, Trim reads based on quality scores, Filter reads based on quality scores, Merge read pairs options will be executed ONLY IF the option is selected.
**Merge read pairs is Paired End only.

More information about the pipeline, including output files and user options, can be found on the Pre-processing QC Pipeline help page

Try it out

You can use either our 16S example files or WGS examples files above to try out this pipeline.

Amplicon Pipelines

Nephele is currently running QIIME v1.9.1, mothur v1.40.5. and DADA2 v1.10. You can use any of these pipelines to run 16S analysis. For ITS, QIIME1 is available.

Each pipeline has different features and steps. Please see the table below to see the different features each 16S pipeline provides in Nephele.

Pipeline Features mothur QIIME1 DADA2
Join forward and reverse short reads as contigs
Screen contigs to reduce sequencing errors
Dereplicate contig sequences
Taxonomic assignment based on selected database
Remove sequences likely due to sequencing errors
Identify and remove chimeric sequences
Classify sequences based on k-nearest neighbor
Remove sequences belonging to undesirable lineages
Remove rare OTUs in the samples
Detect differentially abundant features in samples
Construct phylogenetic tree
Calculate various measures of diversity
Ion Torrent Processing - Beta

More detailed information about each step can be found on the individual pipeline help pages:

Follow our tutorial to try it out.

Metagenome Pipelines

Nephele’s metagenome shotgun pipeline runs the bioBakery Workflows developed by The Hutterhower Lab.

The pipeline runs the Whole Metagenome Shotgun (wmgx) and Visualization for Whole Metagenome Shotgun (wmgx_vis) bioBakery workflows. The features list below are a summary of the Nephele bioBakery pipeline workflow.

Pipeline Features bioBakery
Trim and filter reads for quality with kneadData
Filter contaminant sequences (human and ribosomal RNA) with kneadData
Taxonomic assignment with MetaPhlAn2
Functional profiling with HUMAnN2
Strain profiling with StrainPhlAn
Summary visualizations of species and functional gene abundance
PCoA ordination of species composition
Plots of functional feature detection vs sequencing depth

More information about the pipeline, including output files can be found on the bioBakery pipeline help page.

Try it out

Note: These data are from bioBakery Workflows Tutorial. These files were generated using reads obtained from a set of six healthy and diseased samples (Alterations of the human gut microbiome in liver cirrhosis. Nature. 2014 Sep 4;513(7516):59-64.)

File (Type) Size Description
Sequences 30MB Contains 6 single-end samples sequenced on the Illumina MiSeq platform
Mapping File (Excel) 11KB Metadata file used in Nephele submissions that describes samples, treatments, etc. for analysis
*Note: please unzip the file and upload individual fastq.gz files when submitting. See the tutorial for help on submitting a job.
Downstream Analysis Pipeline

You can use biom files from your 16S or ITS pipeline outputs to run the downstream analysis (DA) pipeline. Nephele's DA pipeline uses QIIME 2 to provide sample observation and taxonomic summaries and diversity analyses of an OTU table

Pipeline Features Downstream Analysis
Summarize sample metadata
Calculate alpha diversity measures
Plot PCoA ordination of beta diversity
Plot bar graphs of taxonomic abundance

More detailed information about each step can be found on the Downstream Analysis pipeline help page.