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.
Nephele v2 supports demultiplexed paired-end and single-end FASTQ files. Please see the supported data type per pipeline below.
| Pre-processing QC | QIIME v1.9.1 | mothur v1.40.5 | DADA2 v1.10 | bioBakery | Downstream Analysis (amplicon only) | |
|---|---|---|---|---|---|---|
| Paired End FASTQ | ||||||
| Single End FASTQ | ||||||
| BIOM V1 | Mapping file from your 16S or ITS pipeline output |
If you need help figuring out what type of data you have, please see the FAQ.
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
You can use either our 16S example files or WGS examples files above to try out this pipeline.
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.
| Amplicon | QIIME v1.9.1 | mothur v1.40.5 | DADA2 v1.10 |
|---|---|---|---|
| 16S | |||
| ITS |
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 |
More detailed information about each step can be found on the individual pipeline help pages:
Follow our tutorial to try it out.
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.
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 |
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.