Transcript ppt - UMD Biological

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)
McGrath, 2007
• 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…)
II. differential expression
2002:
low-throughput
• northern blots
• qRT-PCR
high-throughput
• microarrays (once genes known)
Haag et al., 1998
2013:
• RNAseq:
sequence all mRNAs in sample, count reads
RNAseq
XX vs. XO in 5 Caenorhabditis species
selfing worms have:
• smaller transcriptomes
• less sex-biased expression
of remaining genes
Thomas et al. 2012
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