My Genome Science 2018 talk on sequencing the Zymo microbial community on the Oxford Nanopore PromethION

1. Assessing ultra-deep, long-read
metagenomics on Oxford Nanopore
PromethION
Josh Quick, University of Birmingham

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

5. PromethION run - log distributed

6. Sequencing speed
Overloading the library can result in depletion of ATP
Ligation efficiency is very high with LSK109 so more sensitive to input

7. Refuel

8. Alignment stats
Species Bases N50 Cov
Listeria monocytogenes 1.13E+11 NA 38444.80
Pseudomonas aeruginosa 2.52E+09 NA 370.46
Saccharomyces cerevisiae 1.07E+09 4771 87.78
Bacillus subtilis 1.39E+09 4850 344.18
Escherichia coli 23229830 4171 4.53
Salmonella enterica 24176038 4389 5.00
Lactobacillus fermentum 5816512 2816 2.77
Enterococcus faecium 600248 4546 0.19
Cryptococcus neoformans 811771 5425 0.04
Staphylococcus aureus 102319 4691 0.04

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

12. Normalised representation
Cryptococcus 50-fold and Saccharomyces >2-fold underrepresented using MPZ
-50

13. LFB only
0.5x Ampure + LFB

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.

16. Data availability
FASTQ, raw signal available for download!
https://github.com/LomanLab/mockcommunity

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