I. defining genomes and their contents 2002: Sanger sequencing technology (few, long reads) EST...

I. defining genomes and their contents

2002: Sanger sequencing technology (few, long reads)• EST projects (random cDNA clones)• cloned gDNA (cosmids, fosmids, plasmids lambda phage)

• initial sequencing of human genome: ~15 years, 100’s of people, $3,000,000,000

2012: Next-generation sequencing technology (huge numbers of short reads)• de novo transcriptome assembly• massively parallel whole-genome shotgun• re-sequence human genome (with poor assembly):

one week, ~ 2 people, ~$3,000 (and falling…)

McGrath, 2007

II. differential expression

2002:low-throughput high-throughput• northern blots• qRT-PCR

Haag et al., 1998

2013:• RNAseq: sequence all mRNAs in sample, count reads

• microarrays (once genes known)

selfing worms have:

• smaller transcriptomes

• less sex-biased expression of remaining genes

Thomas et al. 2012

RNAseqXX vs. XO in 5 Caenorhabditis species

III. genotype-phenotype association & evolutionary history

2002: • identify markers (microsatellites, SNPs, RFLP) • build recombination map• map traits (induced or natural) to ever-smaller interval

2012:• localize induced or clinical mutants by resequencing• GWAS/QTL without map or pre-existing markers (even in wild populations)

Implications for:• medicine• cell/developmental biology• evolution• ecological

Sequence data should no longer be regarded as representative samples of a vast and unknowable whole.

We are now in the era of the finite (and affordable) genome.

How big can you think?

• Which regions of the genome are selected when an aphid switches host, when a fish evolves to live in caves, or when a marmot experiences climate change?

• Which transcripts increase in the cortex as a mouse develops ocular dominance, or in an annelid or a lamprey nerve cord as it regenerates?

• Does Silene have X-dosage compensation between the sexes? [just published!]

• Do chytrid-resistant frogs or drug-resistant Trypanosomes have any gene variants in common?

• What is the recombination map for a bat, a penguin, or a Panamanian fig tree?

• Exactly how many new mutations arise in a single generation of Arabidopsis?

• A facility for next-gen sequencing exists in 5115 Plant Sciences (5th Floor)

• Managed by Suwei (“Sue”) Zhao, with assistance from Kongyi (“Candy”) Jiang

• Suwei (R), Kongyi (L) (with Jerry Regier)

http://www.ibbr.umd.edu/facilities/sequencing