“Recent next generation sequencing results”
MACHADO LABMACHADO LAB
MACHADO LABSPRING 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 TranscriptomeMethods to study the Transcriptome
Variety of methods, Illumina is the most common
DNARNADNARNA
Next Generation Sequence is also used to study gene expressionNext Generation Sequence is also used to study gene expression
The short fragments are mapped to the genomeNumber of mapped reads correlated with expression level
RNA-SeqRNA-Seq
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 selection
Sequencing >24 genomes from Trypanosoma cruzi to characterize history of gene exchange and identify genes under selection
Uses of NGS: PhylogenomicsUses of NGS: Phylogenomics
Uses of NGS: MappingUses 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)
pse bog
x
bog pse
x
F1 x x
Fertile
Sterile
Fertile
D. pseudoobscura vs D. p. bogotana
F1
Fertile
Levi Teitz
Uses of NGS: MappingUses of NGS: Mapping
Sequencing pools of fertile and sterile 5th generation backcrossed males to identify genomic regions associated with hybrid male sterility
Sequencing pools of fertile and sterile 5th generation backcrossed males to identify genomic regions associated with hybrid male sterility
1st instar24-32 h1st instar24-32 h
3rd instar120-128 h3rd instar120-128 h
Pupa (yellow)8-16 h after puparium formation
Pupa (yellow)8-16 h after puparium formationAdult7 day oldAdult7 day old
D. pseudoobscura (4)D. persimilis (3)H1 (♀pse x ♂per)
D. pseudoobscura (4)D. persimilis (3)H1 (♀pse x ♂per)
Uses of NGS: Studying the transcriptomeUses of NGS: Studying the transcriptome
Kevin Nyberg
Kawther Abdilleh
Patterns of sex-bias and expression divergence during development, expression and evolution of ncRNAs, evolution of coexpressed genes and networks
Uses of NGS: Understanding the history and genetic basis of domestication Uses of NGS: Understanding the history and genetic basis of domestication
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
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.
Chromosome
Nucleotide diversity by chromosome (100 Kb windows)Π
per
100
Kb
P < 0.001Every chromosome
ChromosomeSignificant
regions >200Kb >300Kb1 22 4 12 14 23 6 04 16 35 12 16 14 27 15 28 8 09 8 1 1
10 4 011 6 112 17 4 1
Total 142 20 3
Regions with significantly low polymorphism ratios (“ROD”, 100 Kb windows). Empirical distribution used to determine significance (5% tail of distribution)
Uses of NGS: Making dreams come true..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.
Larger populationsare expected toharbor more geneticvariation
H = 4N/(1+ 4N)
Weak correlation between population size and variation
Modified from Nevo et al. (1984)
0
0.002
0.004
0.006
0.008
0.010
0.012
0 0.2 0.4 0.6 0.8 1
Proportion of single foundress broods(pollinating wasps, Pegoscapus sp.)
Nu
cleo
tid
e d
iver
sity
Inbreeding P = 0.0016 P = 0.0109
COI (mtDNA)
Nuclear marker: 1G04
p < 0.00001; p < 0.00001
Population structure can be quantified using:1. Proportion of broods with different numbers of
foundresses2. Genetic data
Uses of NGS: Making dreams come true..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 LABMACHADO LAB