Super basecalling

Vaccitech in house nanopore sequencing, recalling .fast5 files with super accurate guppy.

• Open command prompt: type "command prompt" in the windows search bar.
• Open EPI2ME labs, may have to open docker first.
• Open a tutorial: Click tutorials in the top right, click any of the available tutorials.
• Open super_basecalling.ipynb: File>Open from URL... https://raw.githubusercontent.com/Vaccitech/nanopore/main/super_basecalling.ipynb.
• Make a folder contain all .fast5 files (pass AND fail).
• Run the first code cell: click the arrow in it's top right corner.
• Fill the form with the path of the folder containing the .fast5 files, folder to output fastq files to, guppy config file (based on sample and flowcell). None of these folders can have spaces in their names.
• Click >Enter
• Run the next code cell, check that the printed lines are correct.
• Run the final code cell.
• Copy and paste it's output into command prompt.

Share with Bioinformatics

• Add flowcell reports to output folder: copy and paste all extra reports from original sequencing folder e.g. final_summary_*.txt, report_*.html, barcode_alignment_*.tsv
• Zip the folder with all the .fastq and summary files.

• Upload to bioinformatics server using winSCP.
• Let Bioinformatics know what kind of analysis you'd like run.
• 😎

View multiple BAM files

The nanopore sequencer outputs reads into multiple files; if "Minknow" is used to align these reads, multiple .BAM files are produced. With multiple .BAM files it is harder to get a clear overview. IGV is able to load several .BAM files at once if given a list of files to load.

Highlight the .BAM file you want to open. Shift + right click. Copy as path. Paste the paths into a plain text editer, something like notepad and not word. Delete the quotes " around each path. Save as file-name.bam.list. Load as normal in IGV.

If you have trouble try deleting any empty lines from you .bam.list file. Also test in on some files without spaces in the names/folder names.