“Recent next generation sequencing results” MACHADO LAB

SPRING 2013

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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 Variety of methods, Illumina is the most common DNA RNA DNA RNA

Next Generation Sequence is also used to study gene expression The short fragments are mapped to the genome Number of mapped reads correlated with expression level RNA-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 Uses of NGS: Phylogenomics

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)

psebog x pse x F1 x x Fertile Sterile Fertile D. pseudoobscura vs D. p. bogotana F1 Fertile Levi Teitz Uses of NGS: Mapping Sequencing pools of fertile and sterile 5 th generation backcrossed males to identify genomic regions associated with hybrid male sterility

1 st instar h 1 st instar h 3 rd instar h 3 rd instar h Pupa (yellow) 8-16 h after puparium formation Pupa (yellow) 8-16 h after puparium formation Adult 7 day old Adult 7 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 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

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.

Chromosome Nucleotide diversity by chromosome (100 Kb windows) Π per 100Kb P < Every chromosome

Chromosome Significant regions>200Kb >300Kb Total 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.. 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 populations are expected to harbor more genetic variation H = 4N  /(1+ 4N  )

Weak correlation between population size and variation Modified from Nevo et al. (1984)

Proportion of single foundress broods (pollinating wasps, Pegoscapus sp.) Nucleotide diversity   Inbreeding  P =  P = COI (mtDNA)

Nuclear marker: 1G04  p < ;  p <  

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