“Recent next generation sequencing results” MACHADO LAB

“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