Hertweck and Pires presentation from Evolution 2012 in Ottawa, in the Genomics 7 session.

1. Assembly of repetitive DNA
from genome survey
sequencing: Lessons from
grasses and applications to
non-model systems
Kate L Hertweck (NESCent)
and J. Chris Pires (U of Missouri)
mobilebotanicalgardens.org
Sandwalk.blogspot.com

2. Genome sequencing, large genomes and evolution
●
Genome sequencing is becoming a routine laboratory procedure.
●
The first step in genome analysis is masking repetitive elements (REs),
which may compromise a large portion of a genome.
●
Digging through everyone's genomic junk sounds pretty fun!
●
What determines genome size? Why and how?
Kate Hertweck, Repetitive DNA assembly

3. Genome sequencing, large genomes and evolution
●
Genome sequencing is becoming a routine laboratory procedure.
●
The first step in genome analysis is masking repetitive elements (REs),
which may compromise a large portion of a genome.
●
Digging through everyone's genomic junk sounds pretty fun!
●
What determines genome size? Why and how?
●
Methods in large genome de novo assembly of next-gen data are
improving (Schatz et al 2010)
●
Sanger sequencing in Fritillaria indicates highly divergent TEs
(Ambrozova et al 2011)
●
Low-coverage Illumina sequencing in barley identifies both genes and
novel repeats (Wicker et al 2008)
●
Estimation of genome size and TE content in maize and relatives is
accurate with very short paired-end reads (Tenaillon et al 2011)
Kate Hertweck, Repetitive DNA assembly

4. Transposable elements are relevant to evolution
●
Direct: TE movement can disrupt gene function
●
Links between TEs and adaptation/speciation?
●
Indirect: Increases in genome size
●
Many historical hypotheses about relationships
between genome size and life history (complexity,
mean generation time,
habitat/environment/climate, growth form)
●
Physical-mechanical effects of nuclear size and
mass
●
How does TE proliferation affect plant diversification?
Kate Hertweck, Repetitive DNA assembly

5. Our data
●
Illumina (80-120 bp single end), 6 taxa per lane
●
GSS: Genome Survey Sequences
●
Assembled plastomes, mtDNA genes, and nrDNA genes from less than less
than 10% of the GSS data!
●
Poaceae (family of grasses, model system)
●
Medium-sized genomes
●
well-annotated library of repeats
●
Asparagales (order of petaloid monocots, non-model system)
●
Very large genomes
●
discovery of novel repeats
Kate Hertweck, Evolutionary effects of junk DNA
Repetitive DNA assembly

6. Our data
●
Illumina (80-120 bp single end), 6 taxa per lane
●
GSS: Genome Survey Sequences
●
Assembled plastomes, mtDNA genes, and nrDNA genes from less than less
than 10% of the GSS data!
●
Poaceae (family of grasses, model system)
●
Medium-sized genomes
●
well-annotated library of repeats
●
Asparagales (order of petaloid monocots, non-model system)
●
Very large genomes
●
discovery of novel repeats
Kate Hertweck, Evolutionary effects of junk DNA
Repetitive DNA assembly

7. Methodological approaches
1. Sequence assembly:
●
Ab initio repeat construction: use raw sequence reads to build
pseudomolecules or ancestral sequences
●
De novo sequence assembly: standard genome assembly
methods, screen resulting contigs (MSR-CA)
Kate Hertweck, Evolutionary effects of junk DNA
Repetitive DNA assembly

8. Methodological approaches
1. Sequence assembly:
●
Ab initio repeat construction: use raw sequence reads to build
pseudomolecules or ancestral sequences
●
De novo sequence assembly: standard genome assembly
methods, screen resulting scaffolds (MSR-CA)
2. Annotation method:
●
Motif searching
●
Reference library: current RepBase, 3110 repeats, 98.7% are
from grasses (RepeatMasker and CENSOR)
Kate Hertweck, Evolutionary effects of junk DNA
Repetitive DNA assembly

9. Methodological approaches
1. Sequence assembly:
●
Ab initio repeat construction: use raw sequence reads to build
pseudomolecules or ancestral sequences
●
De novo sequence assembly: standard genome assembly
methods, screen resulting scaffolds (MSR-CA)
2. Annotation method:
●
Motif searching
●
Reference library: current RepBase, 3110 repeats, 98.7% are
from grasses (RepeatMasker and CENSOR)
Class I: Retrotransposons Class II: DNA transposons
LTR TIR
LINE Crypton
SINE Helitron
ERV Maverick
SVA
See my iEvoBio talk about TE databasing and ontology!
Kate Hertweck, Evolutionary effects of junk DNA
Repetitive DNA assembly

10. TE assembly and annotation results: Poaceae
Taxon Genome # reads # scaff- Repeat % % % % % %
size (Mb) olds scaff- LTRs Copia Gypsy SINEs LINEs DNA
olds TEs
rice 389 3.8 2376 1718 72 21 48 0.2 4.4 18
sorghum 735 5.3 2248 2255 67 21 46 N/A 2.9 26
maize 2045 5.1 1324 1197 77 21 56 N/A 1.9 18
Kate Hertweck, Evolutionary effects of junk DNA
Repetitive DNA assembly

11. TE assembly and annotation results: Poaceae
Taxon Genome # reads # scaff- Repeat % % % % % %
size (Mb) olds scaff- LTRs Copia Gypsy SINEs LINEs DNA
olds TEs
rice 389 3.8 2376 1718 72 21 48 0.2 4.4 18
sorghum 735 5.3 2248 2255 67 21 46 N/A 2.9 26
maize 2045 5.1 1324 1197 77 21 56 N/A 1.9 18
●
Previous research: Good TE annotations and copy number estimates in
all genomes
●
Our results:
●
Recovery of all extant superfamilies
●
High sequence similarity between scaffolds and reference
sequences
●
Full length LINEs, SINEs, LTRs; fragmented examples of all
●
Abundance estimation is problematic
Kate Hertweck, Evolutionary effects of junk DNA
Repetitive DNA assembly

12. REs in Core Asparagales
Agapanthaceae Xanthorrhoeaceae
●
Reference library is highly
diverged from scaffolds to be
annotated (much lower sequence
similarity)
●
Caution in interpreting results
●
Large scaffolds of some TEs
●
Many small scaffolds of many TE
superfamilies
●
Comparisons of sister clades
Asparagaceae
Naturehills.com ag.arizona.edu
Kate Hertweck, Evolutionary effects of junk DNA
Repetitive DNA assembly

13. Very large genomes in Core Asparagales
Agapanthaceae Xanthorrhoeaceae
Allioidae
Allium
12.9 Gb
5.1 billion reads
1858 scaffolds
Amaryllidoideae
Scadoxus
21.6 Gb
6 billion reads
Asparagaceae
1336 scaffolds
other (RC, satellite, low
complexity, simple repeats)
% Copia LTRs
% Gypsy LTRs
% LINEs
% DNA TEs
Kate Hertweck, Evolutionary effects of junk DNA
Repetitive DNA assembly

14. Closely related lineages have different results
Agapanthaceae Xanthorrhoeaceae
Aphyllanthoideae
Aphyllanthes
2.7 billion reads
436 scaffolds
Agavoideae
Hosta
4.7 billion reads
1084 scaffolds*
Asparagaceae
other (RC, satellite, low
complexity, simple repeats)
% Copia LTRs
% Gypsy LTRs
% LINEs
% DNA TEs
Kate Hertweck, Evolutionary effects of junk DNA
Repetitive DNA assembly

15. Small genomes contain variation
Agapanthaceae Xanthorrhoeaceae
Lomandroideae
Lomandra
1.1 Gb
4.7 billion reads
1491 scaffolds
Asparagoideae
Asparagus
1.3 Gb
5 billion reads
1977 scaffolds
Asparagaceae
Nolinoideae other (RC, satellite, low
complexity, simple repeats)
Sansevieria
% Copia LTRs
1.2 Gb
% Gypsy LTRs
4.9 billion reads
835 scaffolds % LINEs
% DNA TEs
Kate Hertweck, Evolutionary effects of junk DNA
Repetitive DNA assembly

16. Example: LTR from Hosta
Kate Hertweck, Evolutionary effects of junk DNA
Repetitive DNA assembly

17. So what?
●
Assembly of consensus sequences of TEs from very low coverage
sequence data, even without a close reference library
●
Improve annotation (and assembly) by building a library of lineage-
specific TEs
●
Other parameters for genomic comparisons
●
Abundance estimates
●
Characterize genetic diversity within each element
●
Comparative biology of TEs
●
Does TE proliferation contribute to diversification or shifts in
rates of molecular evolution?
●
Are there common patterns between TEs and life history trait
evolution?
Kate Hertweck, Evolutionary effects of junk DNA
Repetitive DNA assembly

18. Acknowledgements
J. Chris Pires lab (U of Missouri)
Dustin Mayfield
Pat Edger
NESCent (National Evolutionary Synthesis Center)
Allen Roderigo
Karen Cranston
www.nescent.org
Twitter k8lh
Google+ k8hertweck@gmail.com
Kate Hertweck, Evolutionary effects of junk DNA
Repetitive DNA assembly

19. Asparagales results
Taxon Genome #reads Total Nuclear % % % % % DNA
size (Gb) (billions) scaffolds scaffolds LTRs Copia Gypsy LINEs TEs
Hosta N/A 4.7 1084 601 52 6 46 0.5 4
Agapanthus 10.2 1.3 438 176 70 32 40 1.7 3
Lomandra 1.1 4.7 1491 532 68 29 39 7.9 6
Sansevieria 1.2 4.9 835 280 67 27 39 4.3 6
Asparagus 1.3 5.0 1977 646 67 35 32 0.5 10
Scadoxus 21.6 6.0 1336 493 73 24 49 0.2 4
Allium 12.9 5.1 1858 539 65 22 44 0.6 10
Ledebouria 8.6 4.1 2481 771 66 35 32 0.4 5
Haworthia 14.9 4.6 1360 481 75 30 45 0.8 3
Aphyllanthes N/A 2.7 436 248 51 24 23 1.2 10
Dichelostemma 9.1 3.9 1706 584 75 38 37 0.2 7
Kate Hertweck, Evolutionary effects of junk DNA
Repetitive DNA assembly