“Recent next generation sequencing results”
MACHADO LAB
MACHADO LAB
SPRING 2013
…….
…….
Phylogeny reconstruction
Population Genetics
My Sequence output:
Graduate Student
~440 Kb
Postdoc
~850 Kb
Now…
Rice project
~430 Gb
My Sequence output:
Graduate Student
~440 Kb
Postdoc
~850 Kb
Now…using NGS..
Rice project
~430 Gb
(Only 41 “experiments”)
Methods to study the Transcriptome
DNA
RNA
Variety of methods, Illumina is the most common
Next Generation Sequence is also used to study gene expression
RNA-Seq
The short fragments are mapped to the genome
Number of mapped reads correlated with expression level
Uses of NGS: Phylogenomics
Phylogeny of a human pathogen (32 genes)
Carlos Flores-Lopez
Sequencing >24
genomes from
Trypanosoma cruzi to
characterize history of
gene exchange and
identify genes under
Uses of NGS: Mapping
Find genomic regions
underlying phenotypic
differences
NGS: sequence pools to identify candidate regions, or genotype
using NGS-based methods (e.g. RAD tags)
Uses of NGS: Mapping
D. pseudoobscura vs D. p. bogotana
pse
bog
bog
x
pse
x
Levi Teitz
F1
F1
x
Fertile
Fertile
x
Sterile
Fertile
Sequencing pools of fertile and sterile 5th generation backcrossed males to
identify genomic regions associated with hybrid male sterility
Uses of NGS: Studying the transcriptome
1st instar
24-32 h
3rd instar
120-128 h
Pupa (yellow)
8-16 h after
puparium
formation
Kevin Nyberg
D. pseudoobscura
(4)
D. persimilis (3)
H1 (♀pse x ♂per)
Adult
7 day old
Patterns of sex-bias and expression divergence during
development, expression and evolution of ncRNAs,
evolution of coexpressed genes and networks
Kawther Abdilleh
Uses of NGS: Understanding the history and
genetic basis of domestication
Uses of NGS: Understanding the history and
genetic basis of domestication
O. glaberrima
O. barthii
African rice domestication
41 resequenced individuals (~30X)
Identification of candidate regions harboring adaptive
genes associated with the domestication process
Strong directional
selection expected to
cause selective sweeps
in genomic regions
associated with
phenotypes under
selection
Artificial selection during domestication and crop improvement involves
selection of specific alleles at genes controlling key morphological and
agronomic traits, and result in reduced genetic diversity relative to
unselected genes.
Π per 100Kb
Nucleotide diversity by chromosome (100 Kb windows)
Chromosome
P < 0.001
Every chromosome
Regions with significantly low polymorphism ratios (“ROD”, 100 Kb
windows). Empirical distribution used to determine significance (5% tail
of distribution)
Significant
Chromosome regions
1
22
2
14
3
6
4
16
5
12
6
14
7
15
8
8
9
8
10
4
11
6
12
17
Total
142
>200Kb
4
2
0
3
1
2
2
0
1
0
1
4
20
>300Kb
1
1
1
3
Uses of NGS: Making dreams come true..
Addressing questions in non-model organisms with no
genomic resources
Under neutrality a positive correlation between levels of
genetic polymorphism (heterozygosity) and population size
is expected.
H = 4N/(1+ 4N)
Larger populations
are expected to
harbor more genetic
variation
Weak correlation between population size and variation
Modified from Nevo et al. (1984)
COI (mtDNA)
Nucleotide diversity
0.012
0.010
0.008


0.006
0.004
0.002
0
0
0.2
0.4
0.6
0.8
1
Proportion of single foundress broods
(pollinating wasps, Pegoscapus sp.)
Inbreeding
: P = 0.0016
 : P = 0.0109
Nuclear marker: 1G04
0.01
0.009
Nucleotide diversity
0.008
0.007
0.006


Series1
0.005
Series2
0.004
0.003
0.002
0.001
0
0
0.2
0.4
0.6
0.8
Proportion of single foundress broods
: p < 0.00001;  : p < 0.00001
1
Population structure can be quantified using:
1. Proportion of broods with different numbers of foundresses
2. Genetic data
Uses of NGS: Making dreams come true..
Collecting RNAseq data to characterize levels and patterns of
genetic variation across multiple (5-10) species with different
mating system (along a continuum of inbreeding levels) and
across thousands of genes.
“Recent next generation sequencing results”
MACHADO LAB