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Post-basecalling analysis
EPI2ME platform
The EPI2ME platform is a cloud-based data analysis service developed by Metrichor Ltd., a subsidiary of Oxford Nanopore Technologies. The EPI2ME platform offers a range of analysis workflows, e.g. for metagenomic identification, barcoding, alignment, and structural variant calling. The analysis requires no additional equipment or compute power, and provides an easy-to-interpret report with the results. Instructions on downloading are available in the EPI2ME protocol
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Open the EPI2ME using the desktop shortcut.
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If prompted, select a folder containing FASTQ files to be analysed.
If a MinKNOW-generated folder structure is detected in the default input location, this will be shown in the Experiments & samples tab. You can change the default input location in the Settings page (cog icon) under Data sources. The root of the Output folder can be changed and any output from the analysis will be stored beneath that path as folders (named by instance ID). The root output location can be changed in the Settings page or when adjusting the parameters for the individual analysis.
Alternatively, you will be directed to the Folders tab, where you can select your input folder. -
Select the analysis you want to perform.
You can select from the full list of analyses (All tab), or from the Favourites tab. Select FASTQ Clone Validation.
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Adjust the run-time parameters of your analysis.
The parameters available to fine-tune will depend on the type of analysis selected.
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Once all the required fields are filled in, click "Accept & start" to start data analysis.
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Follow the progression of upload and download of read files in the Agent.
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Clone validation workflow report
The key figures from the workflow are presented under the Clone Validation tab in the report.
The summary table shows the number of sequence reads that have been analysed per experimental barcode. These data may be used to identify the samples that may have dropped out of the sequence analysis due to insufficient sequence reads.
For each plasmid assembled a figure of genome annotation is presented. The pLannotate software is used to annotate the polished plasmid consensus sequence. In this figure barcode01 has been assembled into a 3037 bp consensus sequence. The addgene database has been used to identify the annotated genes and plasmid features shaded in blue whilst the csgG protein shaded in red has been identified from the Swissprot database.
In addition to the plasmid figure, the same data are also provided in tabular format. This can be used to identify the precise location of the annotated features.
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Results
The EPI2ME Agent will download the polished consensus sequence in FASTA format to the EPI2ME result directory. If the plasmid sequence contains either the T7 or pRham sequencing primers, the cloned region of insert will also be reported. In the figure the files e.g. barcode01.final.fasta correspond to the complete plasmid sequence and the barcode01.insert.fasta correspond to the cloned region of interest.