2 day introduction course to MRC CLIMB and microbial genomics

1. Introduction to nullarbor
The Milner Centre for Evolution
Department of Biology & Biochemistry, University of Bath
http://www.climb.ac.uk/
http://www.sheppardlab.com/
Ben Pascoe
with Maciej Filocha (Warwick) & Mark Pallen (Warwick)

2. 1. Introduction to nullarbor
2. Setting up nullarbor with test campy dataset
3. Launching your own VM
4. (something for you try later – Shigellosis nullarbor tutorial)
5. Checking your nullarbor output
Introduction to nullarbor

3. Sequencing

4. https://github.com/tseemann/nullarbor
Nullarbor - "Reads to report" for public health and clinical microbiology
http://www.slideshare.net/torstenseemann/bioinformatics-tools-for-the-diagnostic-
laboratory-tseemann-antimicrobials-2016-melb-au-sat-27-feb-2016

5. What is nullarbor?
Per isolate
Clean /trim sequence reads (Trimmomatic)
• Remove adaptors, quality scores
Species identification (Kraken)
• K-mer analysis against KNOWN database
De novo assembly (MEGAHIT/SPAdes)
• Fast, confident genome assembly
Annotation (Prokka)
• Genome annotation
MLST calling
• From KNOWN databases
Resistome (Abricate)
• ID AMR genes from KNOWN database
Variant calling from reads compared to
reference
Per dataset
Core genome SNPs (Snippy – from reads
Phylogenetic trees (FastTree)
Accessory genome (ROARY)
Report generation

6. Workshop isolates
4 Campylobacter isolates
All LAB strains – should all be VERY similar…
Run nullarbor
How similar are the isolates?
Is there an explanation for any difference observed?
Implications
11168 widely-used as lab strain and molecular studies based on this reference strain

7. Campylobacter: background
Sheppard et al. (2009) Clinical Infectious Diseases 48:1072–1078
952
22
42
45
177
682
48
1275
661692
61
206
354
257
1034
574
21
Sheppard et al. (2010) Applied Environmental Microbiology 76, 5269-5277

8. Campylobacter: source attribution

9. Campylobacter: introgression

10. Campylobacter: GWAS

11. Linking phenotypes and genotypes using GWAS:
Asymptomatic isolatesVs Symptomatic isolates
Weights association
compared to relative
position on the tree
Sheppard et al, PNAS 2013; Pascoe et al, Environmental Microbiology 2015; Monteil et al, Microbial Genomics 2016, Yahara & Meric et al,
Environmental Microbiology 2017

12. Development of GWAS for use with bacteria: GWAS within clonal complex
Sheppard et al (2013) PNAS 110: (29) 11923-11927
Cattle isolates Vs Chicken isolates
Pascoe et al (2015) Environmental Microbiology
DOI: 10.1111/1462-2920.13051
Good Vs Bad Biofilm isolates
Previous studies were confined
to single clonal complex:
 Bacteria are clonal – difficult
to associations biased by
lineage effects – inheritance
from common ancestor.
 Accessory genome –
bacterial genomes are all
different sizes!

13. Development of GWAS for use with bacteria: pan-genome GWAS
Symptomatic
Asymptomatic
Paired isolates for pan-genome GWAS
FastML tree of 36 paired isolates (pan-genome)
Reduce false positives
Maintain statistical power
Not confined to single clonal
complex
Zero unmapped words
Mageiros & Meric et al, unpublished; Pascoe et al,
unpublished
Previous studies were confined
to single clonal complex:
 Association weighted
against the clonal frame
(tree)
 Paired isolates from many
CCs.
 Use of reference pan-
genome instead of 1 single
reference genome.

14. Genome-wide association of Campylobacter genetic elements with disease
severity / asymptomatic carriage
Pascoe et al, unpublished
High statistical association:
 glycosylation genes
 Iron uptake
 Motility
*scores for all genes in pan-genome from all 77
isolates – 2,996 genes
 Thousands of ‘this’ in ~3,000
genes!

15. AccessVM usingVM box
Using your ip address:
gambia-1: 137.205.69.151
gambia-2: 137.205.69.153
gambia-3: 137.205.69.154
gambia-4: 137.205.69.155
gambia-5: 137.205.69.156
gambia-6: 137.205.69.157
gambia-7: 137.205.69.158
gambia-8: 137.205.69.159
gambia-9: 131.251.130.226
gambia-10: 131.251.130.227
For all:
User: ubuntu
Password: password123

16. Check we have all the files you need
What do we need?
• Input file: allinput.tab
• Reference genome: al111168.fasta
• Reads from MiSeq: *.fastq.gz
(8 files, 4 isolates)

17. Setup nullarbor
nullarbor.pl --name gambia --mlst campylobacter --ref al111168.fasta --input allinput.tab -
-outdir output --verbose
• Type command to setup nullarbor
• Nullarbor will perform checks and give you command to use to start run:
nice make -j 1 -C /home/ubuntu/gambia/output
• run
• Can also run with ‘no hangup’
nohup nice make -j 1 -C /home/ubuntu/gambia/output &

18. It will run for a couple of hours…

19. Launching your ownVM
https://discourse.climb.ac.uk/

20. Nullarbor output: report example

21. Workshop isolates
Are all four isolates very similar?
Which of the 4 isolates were contaminated?
Which isolate was passaged through a chicken?

22. https://discourse.climb.ac.uk/
Nullarbor tutorials on discourse.climb.ac.uk: Can you run this on your ownVM?