2. Motivation for nanopore metagenomics
To apply to clinical translational projects in Birmingham:
Evaluating faecal microbiome transplants for ulcerative colitis (STOP-COLITIS)
Evolution of the respiratory microbiome in cystic fibrosis (w/Jo Fothergill)
Culture-independent diagnosis of meningo-encephalitis (NIHR)
Enable single contig de novo assembly of genomes from complex samples using long reads:
Phylogenetics on whole genomes
Link genes to chromosomes
Identify strain-level variation (haplotyping)
3. PromethION Slide
Exciting opportunity: PromethION
$2000/flowcell based on 3 packs (12 flowcells)
Twin 10 Gbps fibre
Twin Tesla V100 GPU cards (basecalling)
3,000 channels v 512 on MinION (6x)
Up to 24 flowcells in parallel
4. Mock community
ZymoBIOMICS Microbial Community Standard
Even or log distributed (10^2 to 10^8 cells)
2 ug/prep (even), 220 ng/prep (log)
3 Gram-negative, 5 Gram-positive, 2 Fungi
P. aeruginosa, E. coli, S. enterica, L. fermentum, E. faecalis, S. aureus, L.
monocytogenes, B. subtilis, S. cerevisiae, C. neoformans
‘Cellular’ - but shipped in DNA/RNA Shield (lysis buffer)
Illumina data available from Zymo
9. Limit of detection
Total input 7.5 x10^8 cells with lowest abundance organism S. aureus ~500 cells
(~1 pg)
All organisms detected:
4/10 organisms high enough coverage to assemble
3/10 organisms could give gene presence/absence information
10. Improving metagenomic extraction methods
BB method:
Add sample to bead tube
MPZ method:
Spin down and retain supernatant
Resuspend in PBS and add
Polyzyme
Add Lysis buffer and vortex
Spin down and retain supernatant
MPZ-BB method:
Spin down and retain supernatant
Resuspend in PBS and add
Polyzyme
Add Lysis buffer and vortex
Spin down and retain supernatant
Add Lysis buffer and bead beat 3
x 40s
Combine supernatants
Add Lysis buffer and bead beat 3
x 40s
Combine supernatants
11. MinION runs even community
Extraction N50 (kb) Yield (Gb)
BB 4.4 10.3
MPZ 4.3 10.2
MPZ+BB 4.2 16.7
14. Assembly P. aeruginosa
6.7 Mb
B. subtilis
4 Mb
S. enterica (orange)
5.9 Mb (n=45)
E. coli (red)
5.48 Mb (n=42)
E. faecium
2.8 Mb
S. aureus
2.7 Mb
L. fermentum
1.9 Mb
L. monocytogenes
3 Mb
C. neoformans
18.5 Mb (n=263)
minimap2 & miniasm
S. cerevisiae
11.7 Mb (n=69)
15. Future perspectives
Read lengths are relatively short when using bead-beating
Metapolyzyme did not have a big effect on read length and was ineffective for
lysing Cryptococcus
Better methods for long-read microbiome extractions are needed
Yields on PromethION are sufficient for shotgun metagenomics for complex
microbial communities (high inputs are still required)
Genome scale information recovered over a range of 3 logs difference in
abundance, detection over 5 logs.
17. Acknowledgments
University of Birmingham: Nick Loman, Sam Nicholls, Radoslaw Poplawski
University of Vermont: Scott Tighe
Oxford Nanopore Technologies: Divya Mirrington
Cambridge Biosciences: Hannah McDonnell