Plasmid Assembly at the AGRF
Alexis Lucattini
July, 2017
What can you hope to get out of this talk
- Nanopore expectations with low input.
- What plasmids are and why they are useful.
- Applied examples of bioinformatic tools for Nanopore sequencing.
- Potential AGRF partnership with Nanopore sequencing.
List of tools used during this talk.
| Name |
Author |
Focus |
| Poreduck |
A. Lucattini |
Data Handling |
| Albacore |
ONT |
Basecalling |
| Porechop |
R. Wick |
Trimming |
| Canu |
S. Koren |
Assembly |
| Circlator |
M. Hunt |
Assembly |
Plasmid Background
- Plasmids are small circular dsDNA sequences, found mainly in bacteria.
- Bacteria can use plasmids to share genetic information, even between different species.
- Genes on plasmids can encode for virulence factors or anti-microbial resistance.
- But scientists can also use plasmids to clone, transfer or manipulate genes
Traditional Plasmid Sequencing at the AGRF
- Samples sequenced on MiSeq or Sanger.
- Sanger with multiple primers to 'hop' through genome
- MiSeq, huge coverage yet poor assembly.
- Potential market for plasmid length MinION reads.
Nanopore Yields:
You only get out, what you put in (Nutri-grain et al.)
- High molecular weight DNA ( > 1µg) ==> High yields 5-10+ Gbs.
- Low yields in plasmid extraction, sequencing used as confirmation.
- But how low can one go and still achieve their desired outcome?
8 plasmids: 4 bigs, 4 smalls
- Small plasmids retained through clean-ups better than large plasmids.
- Reasons unknown.
- Higher quality DNA?
- Smaller DNA less likely to break?
- Protocol instructs normalise to 700 ng?
- We have a small dilemma…
A handy workaround.
The problem
- Highly unbalanced library.
- Can obtain equal sequencing by lowering all inputs
- Only as good as your lowest yield.
The solution
- Add the good samples once the bad samples have had a go…
- Can use real-time analysis to know when to add.
The real-time analysis pipeline.
