Field-based DNA sequencing can accelerate phylogenomics research. Using portable DNA sequencers, researchers were able to extract, prepare libraries, and sequence DNA from plant samples directly in the field. The field-generated data was of sufficient quality and quantity to identify plant species using BLAST and for informative phylogenetic analysis with minimal processing. This demonstrates the feasibility of real-time field-based genomics and has implications for ubiquitous sequencing and real-time phylogenetic research using mobile sequencing technologies.

1. Using field-based DNA sequencing to
accelerate phylogenomics
Joe Parker
Royal Botanic Gardens, Kew
Department of Zoology, Oxford University
30th November 2016

2. Outline
Intro
-
Real-time phylogenomics
-
Ubiquitous sequencing
-
Implications

3. Images – Wikimedia commons CC BY-SA
(clockwise from top left: Jeroen Rouwkema, @aGastya, author’s own, @RE73)

4. Phylogenetics  Phylogenomics
Stewart et al. (1987):
5 species
1 gene
130 amino acids
2 phylogenies
doi:10.1038/330401a0
Parker et al. (2013):
22 species
2,326 genes
600,000 amino acids
(~700,000,000 simulated)
14 test phylogenies
100 control phylogenies
doi:10.1038/nature12511

5. Real-time phylogenomics
Clockwise from top-right:
Authors’ own; ONT Ltd;
Quick et al. (2016)
http://dx.doi.org/10.1038/nature16996;
Loose et al. (2016)
http://dx.doi.org/10.1038/nmeth.3930
Nanopores…

6. Field-based DNA
sequencing

7. Field-based sequencing: design and questions
Arabidopsis sequencing in
Snowdonia National Park:
Congeneric species with genomes available:
- Arabidopsis thaliana
- Arabidopsis lyrata ssp. petraea
Sample, extract and prepare libraries in the field
Sequence in the field with MinION ‘Rapid 1D’
Replicate lab-based library prep and MiSeq data

8. Field-based sequencing: design and questions
Questions:
Will field-based DNA extraction, library preparation
and sequencing work?
Will the data produced be any good? In any
quantity?
Can we use it for congeneric species ID? By what
method?
What else might we do with the data?

9. Field-based sequencing: Real-time phylogenomics kit

10. Field-based sequencing: Real-time phylogenomics kit

11. Field-based sequencing: Yield and performance
Total field-sequenced yield
>300Mbp (~1.9x)
~80% accuracy
Sequencer yield affected by
reagent conditions and
sample quality as much as by
field conditions

12. Field-based sequencing: ID using BLAST
Assign ID using BLASTN versus reference
genomes; difference statistic calculated e.g.
BLAST 1 database (=true pos): A. thaliana A.lyrata1
A. thaliana A.lyrata1
BLAST2 db (=true neg): A.lyrata A. thaliana A.lyrata A. thaliana
Sequencing platform: ONT 1D ONT 1D MiSeq MiSeq
Total reads: 91,715 25,839 9,476,598 9,659,489
Total hits present DB1 only (TP): 10,322 76 1,078,986 1,491,474
Total hits present DB2 only (FP): 378 2 29,200 12,613
Total hits present in both @ e ≤ 0.0012 22,386 101 4,251,153 3,829,870
Hit Biases3:
Cumulative length 29,636,139 20,424 91,328,759 65,181,756
Cumulative % identities 850,070 119 25,692,193 19,737,356
Cumulative evalues 0.01 (0.00) 0.04 0.02
Mean length 1,324 202.22 21.48 17.02
Mean % identities 37.97 1.18 6.04 5.15
Mean evalues 4.80E-07 -3.89581E-11 1.06E-08 5.46E-09

13. Field-based sequencing: ID using BLAST
Assign ID using BLASTN versus reference
genomes; difference statistic calculated e.g.

14. Field-based sequencing: ID using BLAST
Assign ID using BLASTN versus reference
genomes; difference statistic calculated e.g.
MinION
MiSeq
~50%
accuracy

15. Field-based genomics: assembly and annotation
Species Arabidopsis thaliana A. lyrata ssp. petraea
Data
MiSeq,
300bp
MiSeq + MinION MiSeq Hybrid
Assembler Abyss hybridSPAdes Abyss hybridSPAdes
# contigs 24,999 10,643 37,568 85,599
Largest contig 89,717 413,462 101,114 38,313
Total length 106,455,313 119,031,074 151,562,895 117,256,694
Reference length 119,667,750 119,667,750 183,707,801 183,707,801
N50 7,853 48,730 9,605 1,686
Unaligned length 7,121,882 6,737,059 36,669,847 35,287,390
Genome fraction (%) 82.0 88.7 53.4 43.7
Duplication ratio 1.01 1.06 1.17 1.02
# N's per 100 kbp 1.72 5.41 0.22 7.09
# mismatches per 100 kbp 518 588 1,297 1,097
# indels per 100 kbp 120 130 334 271
Largest alignment 76,935 264,039 44,515 17,201
Total aligned length 98,382,255 108,085,473 100,502,092 80,814,492

16. Field-based sequencing: Phylogenomics
Coding sequences and proteins
predicted from hybridSPAdes
MiSeq + MinION (lab- and field-
sequenced) data assembly using
CEGMA
Aligned to 248 CEGMA alignments
(plus additional plants from JGI)
with muscle
Quick RAxML phylogenies;
TreeAnnotator majority-rule
consensus Consensus support > 98%

17. …. And extreme phylogenomics

18. Field-based sequencing
Conclusions
Field-based DNA extraction, library preparation and
sequencing are entirely feasible.
With current techniques, comparable quantities of
data can be produced to lab-based runs.
Data is of sufficient quality for congeneric ID using
simple, fast processes (BLAST) and genomics.
Raw reads can even be used for informative
phylogenomics with minimal processing.

19. More adventures in real-time ID: Kew Science Festival 2016
Fast ID-by-sequencing:
Generate genomic data for
BLAST identification:
rapid-rough-reference (‘R3’)
Field-sequence unknown
(blinded) samples from panel
Use BLAST triggered by watch
daemon to instantly identify new
reads
Recompute sample ID in real-
time using difference
statistics

20. More adventures in real-time ID: Kew Science Festival 2016
All six species identified
correctly from blinded samples.
Fastest time-to-ID:
20 minutes
R3 databases:
Samples: 6
Avg yield: 32K reads / 46Mbp
N50: 7.4Kbp
12-48h sequencing
Science festival IDs:
6 samples, 3 days
Avg yield: 23K reads / 33Mbp
N50: 2.9Kbp

21. Implications of
ubiquitous
sequencing in
real-time

22. App store informatics

23. Real-time phylogenomics
Informatics:
Field-based sequencing
Real-time analyses
Asynchronous
computation
Phylogenomics:
Metrics on ‘tree space’
Relaxing orthology
Neutral models of
genomic evolution

24. Big genomic data for macroevolutionary questions
What is the tree of
life?
Is ‘sequence space’
constrained – is
evolution reproducible
(‘replay-the-tape’)?
Are higher clades
‘real’?
Why species?
Genomes?
Individuals?

25. Images – Wikimedia commons CC BY-SA
(clockwise from top left: Jeroen Rouwkema, @Nelsonramirezdearellano, author’s own, @soerfm)

26. Thanks
RBG Kew:
Alexander S.T. Papadopulos (@metallophyte)
Andrew Helmstetter (@ajhelmstetter)
Dion Devey, Robyn Cowan
ONT:
Dan Turner, Richard
Ronan, Gerrard
Coyne